qRT-PCR with Standard Curve
- a standard curve using "bulk" RNA from either WT cells (29-13, e.g.) or -tet cells.
- an H2O control containing primers, SYBR green, and H2O.
- a -RT control. Do an -RT for each sample (e.g., -tet and +tet), but it is OK to do it only on the housekeeping gene (e.g., B-tubulin) and not the gene of interest.
Clean your bench and pipetman with RNase Away.
Anywhere below that says H2O means DEPC H2O.
For each gene you are testing, you will also do:
DNase Treatment: with Ambion DNaseI DNAfree kit
- 10μg RNA
- 5μl 10X DNaseI buffer
- 1μl rDNaseI
- 0.5μl RNase inhibitor
- H2O to 50μl
Incubate at 37°C 30 min, add 1μl rDNaseI, and incubate 37°C 1 hr. Add 150μl H2O followed by 200μl phenol/chloroform/IAA (acidic pH). Spin 25 min at 4°C and take top layer. Precipitate RNA with 20μl 3M NaOAc + 1μl glycogen + 800μl cold 100% EtOH in -20°C overnight. Spin down RNA at 13,000rpm 30 min, remove supernatant, dry pellet 10 min, and resuspend pellet in 20μl DEPC H2O. Spec RNA.
Reverse Transcription Reaction:
There are two different kits/protocols that can be used. Applied Biosystems Taqman kit (Brianna) or BioRad iScript kit (Kyle). Both are explained below.With Applied Biosystems Taqman kit
- 4μg RNA per reaction
- 5μl Taqman RT buffer
- 11μl 25mM MgCl2
- 10μl dNTP mix
- 3.5μl Random Hexamers
- 0.5μl RNase Inhibitor
- 1.5μl Multiscribe RT
- H2O to 50μl
Incubate at 25°C for 10 min, 48°C for 45 min, 95°C for 5 min, cool on ice, then add 1.5μl RNaseH (Ambion/Applied Biosystems), incubate at 37°C for 20 min, then at 65°C for 20 min.With BioRad iScript cDNA synthesis kit
- 1μg RNA per reaction
- 4μl 5X iScript reaction mix
- 2μl primer (oligo d(t), random hexamer, etc.)
- 1μl iScript RT
- Depc H2O to 20μl
Incubate at 42°C for 90 mins, 85°C for 5 mins, hold at 12°C
Assume a 1:1 conversion of RNA into cDNA. For example, if you started with 4μg of RNA and ended up with 50μl of cDNA, then you assume cDNA = 80ng/μL.
- Bulk cDNA:
- - RT dilute 80ng/μL at 1:5 = 16ng/μl
- + RT serial dilute
- 1X = 1:5 dilution from 80ng/μL = 16ng/μL
- 1/4X = 1:4 dilution from 16ng/μL = 4ng/μL
- 1/16X = 1:4 dilution from 4ng/μL = 1ng/μL
- 1/64X = 1:4 dilution from 1ng/μL = 0.25ng/μL
- 1/256X = 1:4 dilution from 0.25ng/μL = 0.0625ng/μL
- 1/1024X = 1:4 dilution from 0.0625ng/μL = 0.015625ng/μL
- Sample cDNA:
- Dilution for gene of interest use 1:20 dilution of sample RT at 80ng/μL = 4ng/μL
- Dilution for housekeeping (control) gene use 1:20 dilution of sample RT at 4ng/μL = 0.2ng/μL
- Primers (all primer stocks at 100μM):
- Add 15μL of each forward and reverse primers to tube and dilute with 970μL H2O, primer mix = 1.5μM
Following plate map (example in Q-PCR plate map file) add 2.5μL of cDNA to each well. Mix 1.5μM primers with SYBR green Master Mix in a tube (10μl primers to 12.5μl SYBR green per well for as many wells as needed) and add 22.5μL of primer/SYBR mix to each well. Cover plate with adhesive cover, use squeegee to press down cover, and place in machine.Use program:
- Cycle 1, Step 1 = 50°C 2 min
- Cycle 2, Step 1 = 95°C 10 min
- Cycle 3 40X, Step 1 = 95°C 15 sec, Step 2 =60°C 1 min (take measurement)
- Cycle 4 80X, Step 1 melt curve = 55°C 10 sec, then temp change in 0.5°C increments to 94.5°C (take meas.)