RT-PCR
RT-PCR for Primary Cultured Neurons
A. RNA Extraction
1. Grow neurons in 12-well plate at a density of 0.5 to 1.0 million per well.
2. After treatment, aspire medium and add 250 ul Trizol (Invitrogen) to each well. Mix well quickly, and pipet up and down 5 times with filtered 1 ml tip. Note: the solution will be sticky because of the DNA release, and do not pipet too hard to break the DNA.
3. Freeze at -80 for at least 1 hr.
4. Thaw and add 50 ul chloroform, shake 10 to 20 sec by hand (rotating up and down, not too vigorous).
5. Keep at room temperature for 10 min, then spin at 13,000 rpm for 10 min (in cold room) and transfer the upper phase (yield is about 110 ul, colorless) to a new tube (RNAse free).
6. Add 125 ul isopropanol, mix, keep at room temperature for 10 min, and spin at 13,000 rpm for 15 min (in cold room).
7. Use a pipet (200 ul) to remove the supernatant (usually a pellet should be noticed at the bottom).
8. Wash with 250 ul 70% (or 75%) EtOH, vortex, and spin at 8,000 rpm for 5 min (in cold room).
9. Discard supernatant (again remove by pipet tips), air dry (normally 10 to 15 min until the opaque pellet turns to transparent or no liquid is present), and add 30 ul DEPC water. Incubate at 60 degrees to for 10 min to dissolve RNA. Shaking or flicking during the incubation will help dissolving.
10. Mix 5 ul RNA with 495 ul DEPC water, read OD at 260 and 280. The ration of A should be 1.8 (but we normally got 1.4).
11. Determine the concentration. 1 OD at 260 nm indicate a concentration of 20 ug/ul. The concentration will be the reading times 4. Normally the concentration is about 0.3 ug/ul.
B. RT.
1. Take 1 ug total RNA, mix with 1 ul Oligo dT (0.5 ug/ul), 1 ul dNTP (10 mM), and water in a final volume of 10 ul.
2. Incubate at 65 degrees for 5 min, then chill on ice for at least 5 min.
3. Make 10 ul RT mix: 4 ul MgCl2 (25 mM), 2 ul DTT (0.1M), 2 ul 10X buffer, 1 ul Superscriptase III, and 1 ul RNaseout.
4. Add the RT (10 ul) mix to the RNA/OligodT solution.
5. Incubate at 50 degrees for 50 min, and 85 degrees for 5 min, chill on ice for at least 5 min.
6. Add 1 ul RNaseH, and incubate at 37 degrees for 20 min.
All reagents are from SuperScript III First strand synthesis kit (Invitrogen, Cat 18080-051).
Use program "BINGRT1". Half reaction will be fine, and saves reagent.
C. PCR for BDNF and GAPDH
For 20 ul reaction:
1 ul TR reaction
2 ul 10X buffer
1.2 ul MgCl2 (25 mM)
0.16 ul dNTP (25 mM)
1 ul primer A (10 uM)
1 ul primer B (10 uM)
0.3 ul Taq (1.5 units, Primega, Cat#M186A or 186E)
13.34 ul water
PCR:
94 degrees for 3 min
94 degrees for 30 sec
55 degrees for 25 sec
72 degrees for 30 sec
72 degrees for 5 min
Perform 19 or 20 cycles for GAPDH
Perform 26 cycles for Exon III
Perform 29 cycles for Exon IV
Perform 31 cycles for Exon II
Perform 33 cycles for Exon I
Use program: "BDNF 1"
Protocol for Hippocampal slices RT-PCR
1. Prepare Slices:
Cut hippocampal slices in 400 uM, usually 4 pieces from one hippocampus. Let the slices rest in recording ACSF for 1 hour or longer (need >100 ml ACSF). Switch to ACSF supplemented with or without 40 mM KCl for a defined length of time. then switch to normal recording ACSF for 5-10 minutes.
2. RNA extraction and precipitation:
Transfer treated slices (4 pieces) into one RNase-free Eppendorf 1.6 ml tube. Homogenize by grinding into 250 ul Trizol (500 for half hippocampus). Pipette up and down until no visible debris. Keep at room temperature for 5-10 minutes. Freeze at -80C (or -20C) or continue to next according to the procedures in the Trizol manual:
Add 50 ul chloroform, mix by shaking for 10-20 sec), keep at room temperature for 10 minutes, spin at 4C 13000 rpm for 15 minutes, discard supernatant. Wash with 250 ul 70% ethanol in PEPC H2O and vortex several times. Spin at 4C 8000 rpm for 5 minutes, discard supernatant. Air dry the RNA pellet at room temperature for 5-10 minutes. Dissolve in 50 ul DEPC-H2O at 60C for 10 minutes. Cool and spin down the sample. Take 5 ul and add to 495 ul DEPC-H2O. Measure the OD at 260/280 nms.
Usually the yield from 4 pieces of Hippocampal slices is 5-10 ug total TNA.
3. RT-PCR
Use 1 ug total RNA as template for RT in total volume of 20 ul reaction. Follow RT protocol.
For 20 ul reaction:
1 ul cDNA
2 ul 10x Buffer
1.2 ul MgCl2 (25 mM)
0.16 dNTPs
1 ul for primer (10 mM)
1 ul rev primer (10 mM)
0.3 ul Taq polymerase
13.34 ul ddH2O
PCR:
94 degrees for 3 min
94 degreese for 30 sec
55 degrees for 25 sec
72 degrees for 30 sec
72 degrees for 5 min
4 degrees hold
Repeat 33 cycles for Exon I
Repeat 31 cycles for Exon II
Repeat 26 cycles for Exon III
Repeat 29 cycles for Exon IV
Repeat 20 cycles for GAPDH
Use PCR program BDNF1
Keywords
Adenylyl cyclase (AC), amyloid precursor protein (APP), Alzheimer’s disease (AD), brain derived neurotrophic factor (BDNF), cAMP, cAMP-responsive element (CRE), CREB, extra-cellular regulated protein kinase (ERK), FE65, long-term potentiation (LTP), long-term depression (LTD), memory formation, neuroplasticity, local protein synthesis.