cDNA Reagents
M-MLV Reverse Transcriptase
Gibco-BRL cat no. 28025-013 order from Invitrogen
5 x buffer and 0.1M DTT is supplied with the Reverse Transcriptase
Random Primers pd(N)6
From Amrad Pharmacia cat no. 27-2166 50 A260 units
Dilute to a final conc of 0.1mg/ml for cDNA synthesis.
[Random primer stock solution is 1mg/mL.
Rnasin
Promega Recombinant Rnasin Ribonuclease Inhibitor
Cat no. N2511 2500 units
dNTPs
From Amrad Pharmacia cat no. 27-2035.
Each nucleotide is supplied as a 100mM solution in H20. To make stock dNTPs add
250ul of dCTP, dTTP, dATP and dGTP to ?mls H20. Final concentration is 10 mM
dNTPs. Aliquot 500ul/tube, store at –20oC.
We use GIBCO-BRL sterile, Rnase-free H20 for all our cDNA/PCR work.
RNA stored in H2O not SDS as SDS will inhibit cDNA synthesis.
cDNA Mix
5 x buffer
DTT
Rnasin
DNTPs (10 mM)
Random Primers
Reverse Transcriptase
Total
ul per tube
2.0
0.33
0.33
1.0
1.0
1.0
5.66 µL
Add 1ugRNA+H2O to make the volume up to 10ul. Incubate in 37oC waterbath for 1
hr, then add 15 ul H20. Store at –20oC (or –80oC for long term storage). Use 1 ul per
PCR reaction.
MDR1 Real Time Assay for gene Expression
MDR1 Primers
3065F
3161R
MGB probe
96 bp product
5’-GGACAAGCACTGAAAGATAAGAAAGA-3’
5’-CCTGAGTCAAAGAAACAACGGTTC-3’
5’-AAGTTTTCTATTGCTTCA-3’
B2M
273F
5’-ACTGGTCTTTCTATCTCTTGTACTACACTGA-3’
406R
5’-TGATGCTGCTTACATGTCTCGAT-3’
probe
5’-TGCCTGCCGTGTGAACCATGTGAC-3’
134 bp product
Primers have been optimised at the following concentrations:
MDR1 F and R primers: 300nM
B2M F and R primers: 100nM
MDR1-MGB-fam probe: 200nM
B2M-Taqman-vic probe: 200nM
PCR Mix
25ul/reaction ie well
1X
2xTaqman Mastermix
MDR1 3065F 1/83.3 diln of 100ug/ml
MDR1 3161R 1/83.3 diln of 100ug/ml
B2M 273F 1/250 diln of 100ug/ml
B2M 406R 1/250 diln of 100ug/ml
MDR1-MGB-fam 1/500 of 100uM
B2M-vic 1/500 of 100uM
H20
12.5ul
.30
.30
.1
.1
.05
.05
10.6
Add 24uL mix/well + 1ul cDNA
Standard Applied Biosystems real time cycling conditions.