RT-PCR Procedure using frozen cells From Biotechniques:(1996)
21:1094-1100
Stocks:
Freezing Solution: 0.15 M NaCI + 10 mM Tris, pH 8.0
2x RNase Inhibitor Stock: Add 2 uL of RNase inhibitor to
18 uL of 0.15 M NaCI + 10 mM Tris (pH 8.0) + 5 mM DTT(store at
-200C in 10-uL aliquots). (This solution should not be repetitively
frozen and thawed see Table 2.)
A-mixture:Pre-mixed, aliquoted and frozen solution that
contains all reagents for the RT step except primers. (In this
study, RNase inhibitor was deleted from the A-mixture formulation.)
T-mixture:Pre-mixed, aliquoted and frozen solution that
contains all reagents for the PCR step except primers.
Primers:100 ng of sense primer and 100 ng of antisense
primer/50-uL reaction.
Info on making forzen aliquots for RT and PCR step can be
got from Chen.D et al PCR methods Appl. (1993) 2:351-353.
Procedure for a 50-uL RT-PCR:
- 1.Trypsinize and count cells
- 2.Add 105 cells to a culture tube and spin at ca. 400x g
for 4 mm to gently pellet cells.
- 3.Resuspend cells in 0.5 mL Freezing Solution to adjust cell
concentration to
- 2x 105 cells/mL.
- 4.Place 10 uL of resuspended cells in a 0.2-mL microcentrifuge
tube and add
- 10 uL of 2x RNase inhibitor stock. Note: Final cell concentration
is now 105 cells/mL
- 5.Freeze immediately in a pipet tip box (intended for 10-uL
tips) containing 95% ethanol chilled to -70 oC. An empty 10-uL
pipet tip box will allow a 0.2-mL tube to be about 50% submerged
in ethanol.
- 6.To a new 0.2-mL PCR tube add 3.2 uL A-mixture + 0.5 uL
antisense primer (200 nglitL); maintain at 4 oC until
cells are added.
- 7.Thaw cells rapidly in a room temperature water bath, vortex
mix for 10s and spin tubes for a few seconds to collect all liquid
in the bottom of tubes.
- 8.Add desired number of cells to tubes. Bring volume to 10
uL with diethylpyrocarbonate-treated water.
- 9.Place tubes in a PCR thermal cycler preheated to 42 oC;
incubate for 1 h to complete the RT step.
- 10.Add 39 uL of T-mixture and 1 uL of sense primer (100 ng/uL)
to perform the PCR step at a 50-uL final volume.
- 11.Analyze PCR products on a 5% polyacrylamide gel.