FAQ

Contents

  1. 1 Scientific facts and figures about single molecule RNA FISH
    1. 1.1 How do you know that the spots you are detecting are single RNA molecules?  Couldn't they be conglomerates?
    2. 1.2 How do you know you're getting all the RNA in the cell?
    3. 1.3 How do you know your probes are detecting the right RNA?
    4. 1.4 What is the hybridization efficiency of each oligo?
    5. 1.5 Why singly-labeled oligos instead of multiple labels?
    6. 1.6 Why 20mers?
    7. 1.7 How do you know ribosomes are not preventing RNA detection?
    8. 1.8 How do you know secondary structure is not a problem?
    9. 1.9 What is the deal with transcription sites?
    10. 1.10 Can you detect SNVs?
  2. 2 Probe design
    1. 2.1 How does the Stellaris probe designer work?
    2. 2.2 What is the maximum number of oligos I should use?
    3. 2.3 What is the minimum number of oligos?
    4. 2.4 What if I have a short gene?
    5. 2.5 What part of the sequence should I design against?
    6. 2.6 What about lncRNA?
    7. 2.7 What about introns?
    8. 2.8 What about multiple isoforms?
    9. 2.9 What about close paralogs?
    10. 2.10 Can I use probes across species, like mouse probes on human cells?
  3. 3 Imaging
    1. 3.1 If you do this, it should work:
    2. 3.2 Can I use a 40x objective?  20x?  Air objective?
    3. 3.3 Can I use a confocal microscope?
    4. 3.4 I can't see any signal even though I used the 40x scope in my lab or my core facility's fancy confocal microscope!
    5. 3.5 What kind of camera do I need?
    6. 3.6 Can I use a two-photon?  Light sheet microscopy?
    7. 3.7 Can I do super-resolution imaging?
    8. 3.8 What thickness of cover glass should I use?
    9. 3.9 I can't see anything through the eyepiece!  What gives?
    10. 3.10 How long can I image the samples before the signal degrades?
    11. 3.11 Can I use nail polish to seal my samples?
    12. 3.12 What about using the anti-fade buffers?  Which one should I use?  What are their properties?
  4. 4 Hybridization/washes
    1. 4.1 How long do I need to hybridize?
    2. 4.2 What about the formamide concentration?  Should I adjust it?
    3. 4.3 Does the wash time matter?
    4. 4.4 How should I set up my hybridization?
  5. 5 Protocol stuff and troubleshooting
    1. 5.1 How should I go about testing a new probe?
    2. 5.2 How do I know that my signal is specific?
    3. 5.3 Can you combine RNA FISH with immunofluorescence?
    4. 5.4 Can you detect RNA FISH signal via flow cytometry/FACS?
    5. 5.5 What is a good negative control?
    6. 5.6 Can I check whether my RNA and a certain protein of interest colocalize using combined IF and RNA FISH?
    7. 5.7 What's a good cell-cycle marker to use?
    8. 5.8 What's a good cell boundary marker to counter stain with?
    9. 5.9 Can you detect microRNA with this method?
  6. 6 Some species specific tips
    1. 6.1 Yeast
    2. 6.2 Mammalian cells
    3. 6.3 C. elegans
    4. 6.4 Bacteria
    5. 6.5 Drosophila
  7. 7 Tissue sections
    1. 7.1 Does RNA FISH work in tissue sections? 
    2. 7.2 What tissues have you done RNA FISH in? 
    3. 7.3 What about background?
    4. 7.4 Can you stitch together multiple images to make a huge collage?
  8. 8 How do you grow and prepare your cells/tissue for RNA FISH?
    1. 8.1 Adherent mammalian cell lines
  9. 9 Quantification
    1. 9.1 How do I quantify my RNA spots?
    2. 9.2 Why do I have to circle the cells/embryos/regions myself?  Why can't the computer figure it out?
    3. 9.3 What do I normalize to?

Scientific facts and figures about single molecule RNA FISH

How do you know that the spots you are detecting are single RNA molecules?  Couldn't they be conglomerates?

This is a good question, and one that has a variety of answers. Many of the control experiments that Sanjay did are in his excellent Vargas et al. PNAS 2005 paper. One (beautiful, in my mind) experiment that Sanjay did was the following. Hein vitro synthesized a bunch of target RNA and put it in two different tubes. In these tubes, he labeled the RNA with probes, with the RNA in each tube labeled with a different dye (say, red or green). Then he combined the two tubes, so he had one tube with RNA that was either labeled with red probe or green probe, but not both. He then injected these into the cell and observed. If the RNA were forming conglomerates, then you would expect yellow blobs containing both red and green RNA. If they were single molecules, though, you would expect the spots to be either red or green but never both. The latter is what he observed. You might question whether this holds for endogenous RNA, but he expressed that RNA and compared intensities, and it was the same. This means that the endogenous RNA was also single particles. Nice! Definitely caveats to this, and technically it applies only to this RNA, but whatever, I think this is pretty solid.

There are other things you can do. One is to measure the fluorescent intensity of the spots and show that you get a unimodal distribution of intensities. Pretty weak in my mind, because if you had some spots with two RNA and some with one RNA, these peaks would overlap so much that it would probably look like a unimodal peak anyway. But what do I know.

To me, one of the strongest experiments are some new results from Eric Lubeck and Long Cai (Lubeck and Cai, Nat Meth 2012). They use super-resolution microscopy to actually read out a barcode of different colors along a single RNA molecule. Think about how cool that is for a minute! Anyway, it's very hard to imagine that conglomerates of RNA would show anything like that sort of thing. I think Sanjay has some other similar experiments that corroborate this.

How do you know you're getting all the RNA in the cell?

Honest answer: no idea. What we have done to get at this is compare to qRT-PCR data, for whatever that's worth. I think Singer first did this in Femino et al. Science 1998, and Vargas et al. PNAS 2005 has a nice demonstration as well. In those cases, you can try and use absolute standard curves to get an actual average number of RNA molecules per cell via RT-PCR and compare to what you get by molecule counting via RNA FISH. In Vargas et al. PNAS 2005 (Supporting Figure 5), we got a pretty close correspondence, with the numbers coming within 30% of each other. But given all the vagaries associated with RT-PCR (RT efficiency, PCR efficiency measurement error, etc.), I'm sort of amazed this number came out so close. I think others have shown the same thing with RT-PCR, and so I guess that's pretty good evidence (e.g., Lu, J., & Tsourkas, A. NAR 2009; Markey, F. B., Ruezinsky, W., Tyagi, S., & Batish, M. PLOS ONE 2014). Many have shown (e.g., Raj et al. Nat Meth 2008 Fig. 2, Lubeck and Cai Nat Meth 2012 Fig. 3b) that fold changes in RNA counts are similar when comparing RNA FISH to RT-PCR, but I'm not sure what that really tells you about detection efficiency except that it's the same (maybe good, maybe bad) in both conditions.


Some will tell you that you can detect the same transcript with two different probe sets and look for colocalization between the colors. The idea is that if you detect with both colors, that means that your efficiency is high. I don't think that actually makes sense–if you have an RNA that is inaccessible for whatever reason, this control tells you nothing, and if you have an RNA that is accessible, then a single color will probably detect it. This two color colocalization approach is good for specificity, though...

How do you know your probes are detecting the right RNA?

This is where the two color test comes in handy. What you can do is label every other oligo with a different fluorophore (i.e., R,G,R,G,R,G,R,G...). If the signals colocalize, that is pretty good evidence that you're detecting the right RNA, since it is very unlikely that a whole bunch of different oligos are all binding to the same incorrect target. Usually, you don't need to do this, because if you get good signal in a single color, you are almost certainly detecting the right thing. However, if you are seeing bright transcription sites, they could potentially be off targets because even a single oligo can light those up. If you are doing analysis of those sites, you will probably want to check things out this way. Also, lincRNA are very prone to these sorts of issues and you should really check those out with this "odds and evens" approach (we'll have a paper on this soon).

What is the hybridization efficiency of each oligo?

Lubeck and Cai estimated a hybridization efficiency of around 60-70%, and we have seen similar numbers. Hard to know for sure why it's not 100%, but whatever, if you get enough oligos, you'll be fine.

Why singly-labeled oligos instead of multiple labels?

We have found that, for instance, double labeling oligos can lead to greatly diminished signal. We're not 100% sure why, but we're guessing it may be dye-dye quenching, either from the two ends of the same oligo or from oligos bound to neighboring targets on the target RNA. Either way, we've found they're more expensive and don't work as well, often alarmingly so, so we don't use them. Then again, the Singer-style oligos (~50mers) had multiple labels on longer oligos, and so they can definitely work, but they are a bit of a pain to synthesize.

Why 20mers?

Why indeed! We've had success with somewhat shorter oligos, like 17mers, but below that we have seen some loss of specificity/increased background. Longer oligos can work as well, and we've tried up to 30mers with success. But they use up more real estate on the target RNA and do sometimes seem to give more background, presumably due to more opportunity for non-specific interactions. I suppose you could try messing with the stringency of the hybridization and wash conditions, but we have found little need to go to longer oligos, especially since they cost more and allow fewer oligos to bind to the target RNA.

How do you know ribosomes are not preventing RNA detection?

In the Raj et al. Nat Meth 2008 paper, we simultaneously targeted both the open reading frame (ORF) and the 3' untranslated region (UTR) with differently colored probes and saw good colocalization. Ribosomes should bind to the ORF but not the 3' UTR, so if the ribosomes were causing a problem, we would have noticed many more spots with the 3' UTR probes.

How do you know secondary structure is not a problem?

In some of our early experiments (Raj et al. PLoS Bio 2006), we targeted oligos to the PP7 RNA hairpin, which is a very strong secondary structure, and saw great signal. Same for targeting MS2 RNA hairpins. So I'm not so worried about it.

What is the deal with transcription sites?

So the "transcription sites" are bright foci that you often find in the nucleus that look a bit brighter than the rest of the single mRNA spots. Here's an example:

What we think is happening here is that there is a pileup of nascent transcripts that sit around waiting for processing (or for transcription to finish) at the site of transcription itself. We and others have confirmed by combined DNA/RNA FISH that these really are at the site of transcription itself (see Levesque and Raj, Nat Meth 2013). Note that not every cell will show this site of transcription; this is because transcription is inherently pulsatile, with the gene itself switching between transcriptionally active and inactive states. In cycling mammalian cells, you will usually see between 0 and 4 spots–0, 1, or 2 if in G1, 0, 1, 2, 3, 4 in S/G2. Sometimes you can even see a "doublet" after replication; indeed, this is the case in the depicted example, where the upper arrow points to a single transcription site, whereas the bottom arrow points to a doublet (for a total of 3 transcription sites in this cell). The intensity of the transcription sites can vary from being just as bright as a single RNA molecule (in which case you use colocalization with an intron probe to discriminate; see Levesque and Raj Nat Meth 2013) or sometimes as bright as 10-50, but usually they're around 3-10 times as bright as a single RNA. If you really want to be very careful about whether something is a transcription site or not, then we recommend costaining an intron and looking for colocalization between the intron and the exon.

Can you detect SNVs?

For a long time, the answer was no, but Marshall Levesque (awesome former PhD student in the lab) came up with a nice method based on the toehold exchange work of David Zhang. You can read about it here.

Probe design

How does the Stellaris probe designer work?

The probe designer does a bunch of bioinformatic analysis that restricts areas prone to cross-targeting and background.  It also uses some more information we have gathered over the years that tells us about what sequences are more prone to background.  Sorry, can't really divulge more than that for various reasons...

What is the maximum number of oligos I should use?

In our initial paper (Raj et al. Nat Methods 2008), we used 48 oligos, and the implication was the more the merrier.  In some sense, that is true, but we have found that actually we seem to get the best results with around 30 oligos most of the time.  If I had to guess, the reason is that each oligo also adds background, so given our currently probe design rules, 30 seems to be a nice balance between having enough oligos on there while also keeping background reasonable.

What is the minimum number of oligos?

This seems to be fairly target specific.  We have definitely gotten really good signal with around 12 oligos, but have gotten pretty crummy signal with 12 oligos, and crummy signal even with 48 oligos.  Just seems to depend on how well the individual oligos perform, which is hard to predict a priori.  Again, we think 30 is a pretty good sweet spot.

What if I have a short gene?

Well, depends on short.  You might be able to get away with just a few oligos, potentially enabling detection of a transcript that is only a few hundred bases long.  Only thing is that the probe designer may not let you do that because some of the sequences are "bad" for whatever reason.  Ignore the probe designer at your own peril, though–you could end up with a lot of background, although who knows.

What part of the sequence should I design against?

If you're targeting mRNA, stick to the coding sequence if possible.  This is under the most selection pressure and will have the least amount of sequence "contaminants" that can cause trouble.  Sometimes this just won't allow enough oligos, though, in which case, then we start venturing into the UTRs.  Can cause trouble, but with the latest probe design software, it's a bit less of a risk.

What about lncRNA?

For lncRNA, just use the regular probe design software.  You may have to watch out for some off-target effects, though (see troubleshooting).  If you get bright nuclear spots, they might be real, might be not real.  Do the odds/evens approach.

What about introns?

Introns can work just fine.  You will usually get a bright spot at the site of transcription (if the gene is actively transcribing–remember that most genes, even GAPDH, show bursts of transcription).  We try to stick to the most 5' part of the intron and also try not to let the oligos spread out too far (i.e., limit to around a 2-3 kb region).  You might need to go further if too much of the region is untargetable for whatever reason, but that's how we start.  Also, probably best to stick to a single intron, but sometimes you have to spread across multiple introns.  20 oligos will usually do the trick for introns.

What about multiple isoforms?

Well, depends what you want to do.  If you want to make a "pan" isoform probe that targets all the isoforms, we usually use the UCSC genome browser to find the isoform that is common to all the probes and design against that.  If you want to target a specific isoform, well, then you have to make probes against a unique part of the isoform, which can be hard because they're often quite short.  There was a nice RNA FISH paper (Waks et al. MSB 2011) that looked at alternative splicing using our method, but targeted isoforms that had really big differences.

What about close paralogs?

We have some software that we haven't yet made publicly available that can design a set of specific oligos that minimize cross targeting with a desired stringency.  Could be a while before it's available for general use, though.  If you want to give it a try manually, I think you want at least 4 mismatches to ensure that the probe doesn't bind off target.

Can I use probes across species, like mouse probes on human cells?

As a general rule, the answer is no. Even if there is a strong degree of conservation at the protein level, there are typically enough sequence differences at the nucleotide level to prevent the probes from binding–even just a couple base mismatches can make a huge difference. You might get some weak binding, and you might even get some signal, but it won't be very robust and so we strongly recommend just getting different probe set for each species-specific transcript. In the long run, you will be much better off that way.

Imaging

We've gotten a lot of questions about imaging, and this is often a point of failure for many people.  Hopefully this can help answer a few questions that people often have.
What imaging setup should I use?
Well, that's a hard question, since there is so much variety, and many things can work, although not all of them will work, certainly not right away.  But...

If you do this, it should work:

  1. Use a 60x or 100x objective with an NA of 1.3 or higher.  This will be an oil immersion lens.
  2. Use a decent light source.  This means mercury lamp or metal-halide (ExFo or something like that).  I've heard of people saying that the Nikon Intensilight is not quite powerful enough.
  3. Use a cooled CCD camera.  Should be cooled to at least -20C.  Less than that can give background that drowns out the signal.
  4. Use the right filter sets.  Duh.
  5. Make sure all the light filters are out and your light throughput is maximized.  Sometimes, they leave in a neutral density filter here or there or leave an aperture closed up.
  6. You may need longer exposures than you might be used to.  We often use exposures of a couple seconds.

Can I use a 40x objective?  20x?  Air objective?

Not typically.  The issue is that you will not typically get enough light because the numerical aperture of the lens is typically smaller.  You might be able to see overall differences in fluorescent intensity, but you will have to do all the appropriate controls to check if it works.

Can I use a confocal microscope?

The short answer is no.  The longer answer is maybe.  I've definitely seen some people use confocal and get decent images.  Personally, I've only had good luck using a spinning disk confocal, but never managed to get anything real out of a point scanning confocal.  That said, if you know enough about optics to disagree with me, then you probably know enough about optics to get it to work.  If you are just starting, I STRONGLY recommend starting with just a regular widefield fluorescence microscope.

I can't see any signal even though I used the 40x scope in my lab or my core facility's fancy confocal microscope!

See above.  Seriously.

What kind of camera do I need?

We used cooled CCD cameras.  We have found that using a non-cooled CCD typically just has too much background to see the signals.  But you don't need a super crazy cooled camera, nor do you need an EMCCD.  A standard CoolSNAP-class -20C CCD will do the trick.  In fact, we have found that the EMCCDs don't really buy you much, and so are not really worth all the extra expense.

Can I use a two-photon?  Light sheet microscopy?

Thomas Gregor at Princeton does this, I think.  And I'm pretty sure other people are doing light sheet microscopy as well.

Can I do super-resolution imaging?

Well, technically, because our spots are usually spread out, we are already localizing the center of the particle with precision below the diffraction limit (I think this is called FIONA?).  You would only need super-resolution if your spots were so crowded that they were all over the place.  The place to learn about super-resolution RNA FISH is Lubeck and Cai Nat Methods 2012.

What thickness of cover glass should I use?

We usually use #1 cover glass, and that seems to work for us.  Which is weird, because most microscopes are designed for #1.5 cover glass.  Hmm.  We have gotten good signals from #1.5 cover glass sometimes, but I also had some mixed results way back in the day, so I usually just tell people to stick with #1.  Anyway, that's my experience for whatever it's worth.  Further note: we often use the Lab-Tek chambered cover glass for our experiments.  We use the Lab-Tek 1 chambers, which have #1 cover glass.  There's also Lab-Tek 2 chambers, which are like version 2.0 of the Lab-Tek chambers and are superior in every way, except that they use #1.5 cover glass.  So we don't use those.

I can't see anything through the eyepiece!  What gives?

The signals are typically so dim that you can't see them without the aid of a cooled CCD camera.  So you really shouldn't see anything when you look through the eyepiece most of the time.  That said, if you have a nice bright probe and a broad optical filter, we have sometimes been able to see the RNA by eye through the eyepiece.  Which is a particularly satisfying experience...

How long can I image the samples before the signal degrades?

I think Biosearch uses VectaShield or something like that, which can preserve the signals for a long time (i.e., weeks or longer).  We usually just use glucose oxidase or just 2xSSC for our imaging, and find that the signals can degrade over 48 hours.  So usually, we try to image within the first 24 hours after hybridization/washing, and keep the sample at 4C when we're not actively imaging it.

Can I use nail polish to seal my samples?

It can work, but we generally recommend NOT doing this, especially if you trying this out for the first time.  The reason is that nail polish can introduce a lot of background, especially in the Cy3 channel.  What can happen is that the nail polish will wick into the space between the coverglass and the slide and into the sample, causing background.  It is possible for it to work, though, and we do use it sometimes in the lab.  What we do is make sure to squeeze out as much liquid between the coverslip and the slide as possible (wicking away all excess liquid using a Kimwipe or something) and then sealing the sample down with a thin layer at the edge.  This seems to work most of the time, but you really have to be careful to pinch things down as tight as possible beforehand and also avoid imaging near the edges.

What about using the anti-fade buffers?  Which one should I use?  What are their properties?

If we don't need any anti-fade buffers (i.e., we are using Cy3 and Alexa 594, etc.), then we just mount with 2xSSC.  If we are using a dye that bleaches a lot (I'm looking at you, Cy5), we always just use the glucose oxidase solutions described in our Nat Meth 2008 and MIE papers.  However, others have used other antifades (including those with DAPI included in them) and gotten good results.  I've heard good things about some of these like ProLong Gold, but these can often interact in unpredictable ways with specific dyes.  For instance, we have some anecdotal evidence that VectaShield works good with the Quasar 670 dye from Biosearch, but does not help with Cy5 from GE/Amersham.  Also, we've seen that VectaShield can sometimes give a strong background fluorescence when used with Quasar 570 and so you may want to avoid it if possible.  Anyway, overall, we're very happy with the glucose oxidase solution.  It's a bit fussier, but it works well in a variety of situations.

Hybridization/washes

How long do I need to hybridize?

Biosearch recommends doing 4 hours.  Certainly, that's enough to see signals in many cases.  We usually just do it overnight because the signals are a bit brighter, but it will usually give you qualitatively the same answer.  (Overnight hybridization also give you the entire next day for imaging.)  I think a couple people I knew did some test in the past and found that the signals didn't get much better after 12 hours, so that's probably the maximum amount of time you need.  Stay tuned, though–may have some updated guidance on this soon.

What about the formamide concentration?  Should I adjust it?

You can adjust it, but I wouldn't advise deviating from the 10% we recommend.  We have found that if you increase the formamide concentration, you will lose signal, and so you will end up counting fewer RNA than are actually there.  In the end, if you have high background, honestly, tuning the temperature or formamide concentration won't really help you out very much.  Most likely your probes are just suboptimal...

Does the wash time matter?

Well, if you leave it in the wash buffer forever, you will get issues, but it doesn't seem to matter that much.  We do 2x 30 minute washes, but you can do another hour or two without too much trouble.

How should I set up my hybridization?

Well, basically just follow the directions from Biosearch.  One thing that we often do is to put the hybridization solution on the cells and then put another cover slip on top.  That spreads the hybridization solution out evenly and also helps prevent evaporation.  Another thing is that we do our hybridizations at 37C.  This can lead to some drying, even with the cover slip, so we often will take a kimwipe, twist into a little knot, put 1mL of water on it and then put that in the dish along with the hybridization.  The increased humidity can keep stuff from drying out.

Protocol stuff and troubleshooting

How should I go about testing a new probe?

So the first thing is to check out whether the probe itself works.  We usually recommend starting with a cell line that you know expresses the gene.  Repeat: a cell line that you KNOW expresses the gene.  If you see spots, then you know your probe is okay.  Otherwise, could be a sample thing, could be an imaging thing, could be that your cells don't actually express the gene you thought, etc.  If you don't have such a cell line, then you can always clone the gene and express it.  Kind of a pain, but it should work.  We STRONGLY recommend testing in a cell line before trying in tissue.  Tissue can have a host of other problems, and so it's best to know that your probe works before even getting into it.

How do I know that my signal is specific?

Usually, if you see the nice tight discrete spots that are in all the pictures, then your signal is most likely specific.  You might, however, see some background.  Here are some notes:
  1. Non-specific blobs will often look a bit larger than the "tight" spots of a real RNA FISH signal.  They can also look a bit irregular.  Sort of like looking for a funny mole.
  2. Sometimes you will see such irregular blobs in the perinuclear area or in the cytoplasm around the nucleus.  This often is background autofluorescence from cell blebbing, etc.  The easiest way to check if this is the case is to take a picture in a green (i.e., GFP) channel to see if you still see the spot.  Autofluorescence often shows up in the green and is very broad spectrum, and so bleeds through into multiple fluorescence channels.
  3. Sometimes you will see such bright, irregular blobs in the nucleus.  This can sometimes come from a single oligo binding to some weird off target transcriptional foci or something in the nucleus.  Often you'll get this sort of thing when targeting lncRNAs, so be careful!  In this case, the best way to check what's going on is to label every other oligo with a different dye (i.e., label your oligos R, G, R, G...).  Correct signal will colocalize nicely.  Off target signal from a "rogue oligo" will not colocalize between the channels.  Main point is that you should be really careful before overinterpreting bright blobs in the nucleus!
  4. Some probes will give a diffuse nuclear background.  Hmm.  Well, that's just some amount of off-targeting binding in the nucleus, probably to some transcriptional junk.  Usually, this is fairly manageable and doesn't really affect the punctate signal anyway.  If it's really bad, though, well, just have to do the odds/evens thing from point 4 above or just get some new oligos...

Can you combine RNA FISH with immunofluorescence?

The answer is most definitely yes!  You can do this in a number of different ways.  The easiest way, although it doesn't always work, is to just add the primary antibody in with the hybridization overnight and then add the secondary during the second wash.  Doesn't always work, though, because sometimes antibodies are not compatible with the hybridization conditions.  In that case, you can either do the RNA FISH first and then do the IF, or do the IF first and then the RNA FISH.  I would probably do the former.  Just be sure to do the IF relatively quickly, and also try to keep the salt high, which will help keep the oligos on the target RNA during the IF procedure.  Also, try not to use serum as a blocking agent.  We have found that those serums can have a lot of RNases in them, and will often kill off all your RNA FISH signal.

Can you detect RNA FISH signal via flow cytometry/FACS?

Answer is a qualified yes.  The problem is that the signal is often very dim, so it's hard to get any real dynamic range.  For highly abundant RNA (e.g., viral), you can really see it, though.  If you're going to try it, be sure to go in with an unstained control as well as a non-specifically stained control (e.g., stain for GFP even though your cells don't have GFP or something like that).  The latter control will tell you (sort of) how much signal you get from background.

What is a good negative control?

That's tricky.  The question is negative control for what?  If you want to show that your signals are specific, we think the strongest experiment is the odds/evens dual labeling that we talked about earlier in the "How do you know you're detect the right RNA?" section.  You can of course stain with a GFP probe or a scramble probe or whatever, but that's not really a negative control.  Those are just a different set of probes that will stain different background things in the cell.  For instance, you might find some bright non-specific nuclear blobs with your GFP "negative control" that don't show up when you use your actual probe.  What does that mean?  Just means the GFP probes are different, that's all–doesn't tell you anything about what your probes are binding to...

Can I check whether my RNA and a certain protein of interest colocalize using combined IF and RNA FISH?

Well, this is tricky.  In some cases, maybe yes.  But often times, the protein of interest will just give a relatively uniform staining in the nucleus, which makes it very difficult to say whether the RNA and the protein "colocalize".

What's a good cell-cycle marker to use?

We often use Cyclin A2 mRNA, which only shows up in S, G2 and M phase (and a little bit early in G1).  We've validated that this actually happens by doing a Click-iT EdU staining at the same time, which stains cells that are in S phase through pulse labeling of newly incorporated DNA nucleotides (see Levesque and Raj, Nat Meth 2013).  Here's a pic of Cyclin A2 in foreskin fibroblasts where you can clearly see some cells as positive and some are negative.  The positive ones were Click-iT EdU positive.

What's a good cell boundary marker to counter stain with?

Umm... working on it.

Can you detect microRNA with this method?

The issue with miRNA is that they are very short and so you can really only get one oligo on there.  That means you lose all the specificity and signal you gain from the tiling approach we use on longer RNA.  That said, others here at Penn have used LNA and signal amplification to do this sort of detection.  Haven't tried it myself, but the results look really nice.

Some species specific tips

Yeast

This is all courtesy of Lenny Teytelman, who spent a long time working through the details.  Main thing: you really need to watch out that you have completely removed the cell wall with zymolyase, otherwise you can sometimes get "variability" that's actually just variable permeability.  Here's a more detailed protocol.  Again, huge thanks to Lenny for figuring this stuff out.

Mammalian cells

Generally, the protocol works really nicely for adherent cells as long as they are not too tall. Fibroblasts, HeLa, A549–these all work great, along with many cell types. Sometimes you can get some issues with non-adherent cells, though. One way to handle it is to affix them to a coverslip and then do the RNA FISH protocol as for adherent cells. Otherwise, you can instead do the RNA FISH protocol in suspension. Two things to watch out for: sometimes, the cells get stuck to the sides of the tube during spin down wash steps, especially with the formamide-containing wash buffer. We've found that adding a bit of Triton-X (0.1%) can help. The other thing is that if you have thick cells you really need to smush them to get good signals. For example, mouse ES cells will give much better signal if you smash the cells together between two coverslips before imaging.

C. elegans

Two situations: embryos and larvae/adults. In embryos, the main thing is that there's a chitinous eggshell you need to break through. We've found that putting the embryos in fixative and then freeze-cracking with a snap freeze in liquid nitrogen seems to do the trick. Then just let them keep fixing for a few minutes more and you should be good to go. When imaging, just be sure to smush the embryos down a bunch–critical for good signal. For larvae/adults, the most important thing is to smush them down. Adults in particular are really thick and you have to smush them or you just won't see anything at all.

Bacteria

Works fine in bacteria. Main thing is that you have to permeabilize the cell wall. You can usually do this with a mild lysozyme treatment (which is what we did in B. subtilis). You can either do it by sticking them to a slide or by doing it in solution–both work. Ido Golding has a good Nature Protocols paper on this. Quantification is a bit of a hassle, because the cells are so small that even moderate amounts of expression can lead to overlapping RNA spots. You can use total fluorescent intensity, though, which is a decent proxy.

Drosophila

More coming...

Tissue sections

Does RNA FISH work in tissue sections? 

Yep, it works quite nicely. Check out these pics. There are a couple of considerations when doing RNA FISH in tissue, though, especially if you're doing it for the first time:
  1. Start with fresh frozen tissue rather than formalin-fixed paraffin embedded (FFPE), which often has more background and is more problematic. We have definitely gotten good results in FFPE, but I think it depends a lot on the sample quality, which tends to be more variable with FFPE. Main thing is to try and make sure your samples went into the deep freeze or the cross-linker shortly after tissue extraction. The longer it waits around, the more the RNA will degrade.
  2. Try and keep short fixation times. For fresh frozen, you can fix the slice in 10 minutes with 4% formaldehyde/1x PBS just like regular adherent mammalian cells. Longer fixation can increase background.
  3. Make thin slices.  We try and use 5µm slices, and thicker than 10µm can sometimes cause background problems.  Not necessarily, but it can.
  4. Try to section onto charged cover slips (just dip in poly-lysine for a bit) rather than slides. The issue is that the imaging typically works best when there is a minimal distance between the cover slip and the sample itself. If you do feel compelled to put your sections on slides, then be sure to wick out as much liquid as possible once you put the cover slip on so that it hugs the sample as tightly as possible.
Also, check out Shalev Itzkovitz's excellent Nature Protocols paper for some other considerations.

What tissues have you done RNA FISH in? 

We've successfully done mouse lung, colon, liver and embryos, and human lung and colon. At least that's what I can remember. Probably done some others as well. It works nicely in most tissues, as far as we can tell.

What about background?

Tissues are certainly more prone to background than other sample types. We definitely recommend taking pictures in the unstained GFP channel (as mentioned earlier in this FAQ) to check for autofluorescence, which usually shows up in all channels. The background can often be very bright and so contrasting can sometimes hide your signal, so be sure to look carefully at your pictures.

Can you stitch together multiple images to make a huge collage?

Yes, and it's super fun. Check out this example of LGR5 in a mouse adenoma.

How do you grow and prepare your cells/tissue for RNA FISH?

Adherent mammalian cell lines

These are usually the easiest.  We typically use chambered coverglass from Lab-tek.  These are very handy because you can grow your cells directly in the chamber and then do all your fixation, hybridization, washing and even imaging right in the same well.  Note that they also have a version 2 of this product, but we have honestly had less success with that product than with the original Lab-tek chambered coverglass.  We usually get the two well chambers because you can slip a 18x18mm coverslip on top of you hybridization solution in order to spread out the solution and reduce evaporation, but the other sizes also work just fine.

Quantification

How do I quantify my RNA spots?

We recommend using our RNAquant software: http://rajlab.seas.upenn.edu/StarSearch/launch.html

We wrote this software largely with the same algorithms that we use in house for quantifying spots.  Hopefully it's pretty self-explanatory.

Why do I have to circle the cells/embryos/regions myself?  Why can't the computer figure it out?

Well, turns out that computers are pretty bad at that sort of thing.  It's often very hard to get the computer to reliably assess where one cell ends and the next begins.  Although it may be possible in some situations, it often requires a ton of work to make it robust, and the resulting code is seldom useful in other contexts.  So the most efficient method we've found overall is to just circle cells by hand.  But if you know of a better way, let us know!

What do I normalize to?

Part of the beauty of having absolute molecule counts is that you don't have to normalize.  You get absolute numbers!  But you may also want to stain against GAPDH or something at the same time just to make sure your RNA FISH worked (and to make sure the cells aren't dead or something like that).  Then again, there are some fundamental questions here about what the right number to report is.  Stay tuned...




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