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RT-PCR Analysis
Solutions
10X RT Buffer
10X PCR Buffer
100 mM Tris pH 9.0
500 mM KCl
1% Triton X-100
25 mM MgCl2
use at a concentration of 1.5 mM
Lysis Solution
4M GuSCN 250 g guanidine thiocyanate
25mM Na citrate 7.0 17.6 ml 0.75M Na citrate pH 7.0
0.5% Sarkosyl 26.4 ml 10% Sarkosyl
add 293 ml Q
before use, add 72ml bME
Water Saturate Phenol
thaw 500 ml phenol
add 0.5 g hydroxyquinolin
add 500 ml Q
mix and allow phases to separate at room temperature
repeat 2 times and store at 4°C
3M NaOAc 5.2
24.6 g NaOAc (anhydrous)
pH to 5.2 with acetic acid
up to 100 ml Q
Procedure
rna isolation
• Add tissue to 400 microliters lysis solution with bME added just prior to use. If collecting several samples hold tubes on ice. These can be stored for years at -80°C.
• Add 1 microliter glycogen, 30 microliters 3M NaOAc 5.2 and 500 microliters water saturated phenol. Mix by gentle inversion and add 100 microliters chloroform.
• Mix by inversion and hold on ice for 15 minutes.
• Spin for 10 minutes at 4°C and remove the aqueous phase. Add 500 microliters isopropanol, hold at -20°C for 1 hour and spin at 4°C for 30 minutes. Wash and dry.
• Resuspend the pellet in 300 ml Lysis Solution and add 300 ml isopropanol. Hold at -20°C for 1 hour and spin at 4°C for 30 minutes. Wash and dry.
• Resuspend the pellet in 10 ml DEPC treated Q and store at -80°C.
reverse transcription reaction
• Add 5 microliters RNA to a sterile siliconized eppendorf tube along with 1 microliter of oligo dT (0.25 mg/ml).
• Heat to 65° for four minutes and immediately transfer to ice.
• Quickly spin the RNA/oligo dT and add 14 microliters of the RT cocktail:
2 microliters 10X RT buffer
1 microliter 1 mg/ml BSA
1 microliter 20 mM DTT
0.5 microliters RNaseIN
0.4 microliters 25 mM dNTPs
0.2 microliters AMV RT
9 microliters Q
• Incubate at 48° for 30 minutes, dilute 5 fold and store at -20°.
pcr reaction
• Dilute cDNA (5 fold) and use between 1-5 microliters of cDNA per PCR reaction.
• Add the following PCR cocktail per tube:
2.5 microliters 10X PCR buffer
1.5 microliters 25 mM MgCl2
1 microliter each primer (10pmol/microliter; 55° Tm)
0.2 microliters 25 mM dNTPs
0.025 microliters a-32P dATP
0.02 microliters Taq polymerase (protocol T.2)
14 microliters Q
• Perform PCR using the following cycle parameters:
94° for 4 minutes, (94° for 30 seconds, 55° for 30 seconds, 72° for 30 seconds) x n, 72° for 5 minutes.
• The number of cycle should be determined empirically to find the linear range of amplification.
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