Xbp1 splicing (EvA lab)

XBP1 SPLICING ASSAY v.2015

STEP 1. RNA extraction (TRIZOL or IsolRNA reagent)

- To stay on the safe side, allow one 6w of 80% confluent cells per sample. It is possible to extract enough RNA from a 12w but the risk of losing the RNA pellet is much higher

- Put cells on ice

- Aspirate medium and add 500 µL of Trizol/Isol straight into the well

- Incubate 5 min on ice

- Recover in eppendorf. Freeze at -80ºC or extract immediately as follows

- Add 100 µL of chloroform, mix well, leave 3 min at RT

- Spin 15 min at 11,000g (about 10,000 rpm in tabletop centrifuge) at 4ºC

- Recover upper phase (transparent), discard lower phase (pink)

- Add 250 µL of isopropanol, leave10 min at RT (or on ice for longer)

- Spin 10 min at 11,000g at 4ºC.

- Wash pellet with 1 mL of 75% ethanol

- Spin 5 min at 7,000g, at 4ºC

- Dry RNA pellets

- Add 50 µL of RNAse-free water. Pipette briefly.

- Incubate 5 min at 55°C, pipette again until dissolved

- Determine RNA concentration with nanodrop. Usually: 100-1000 ng/µL

STEP 2. Retro-transcription

- Retrotranscribe 2 µg of RNA

- If you use Promega RT polymerase, set up 20 µL reactions as follows:

5x Buffer 4 µl

Mg 2.4 µl

dNTPs 10mM each 1 µl

oligos Random/dT 0,5µg/µl 1 µl

Rnasin 0.5 µl

RT polymerase 1 µl

RNA 2 µg = ? µl

H2O to 20µl

- Cycle: 10’ at 25°C à 60’ at 42°C à 10’ at 70°C

STEP 3. PCR amplification

- PCR 2 µL of cDNA

- if you use GoTaq Flexi G2 (NB: Flexi needs Mg), set up 50µl reactions as follows:

H₂O 30,75µl

5x green GoTaq Buffer 10µl

dNTPs 10 mM each 1µl

Mg 2µl

Fwd primer (10µM) 2µl (hXBP1.3S or mXBP1.3S)

Rev primer (10µM) 2µl (XBP1.12AS) see below

GoTaq pol 0,25µl

cDNA 2µl

- PCR cycle:

95°C 2’

95°C 15” |

64°C 30” | 35 cycles

72°C 30” |

72°C 8’

- Primers are from D.Ron:

Fwd human hXBP1.3S AAA CAG AGT AGC AGC TCA GAC TGC

Fwd mouse mXBP1.3S AAA CAG AGT AGC AGC GCA GAC TGC

Rev (hum & mou) mXBP1.12AS TCC TTC TGG GTA GAC CTC TGG GAG

STEP 4. Electrophoresis

- Run 20 µl of the PCR reaction on a 3% agarose gel

- PCR products are 473-457 bp for human, 476-460 bp for mouse. An (annoying) hybrid band runs just above the unspliced form.

- Run slowly and as long as possible to better separate the forms