dnase消化后逆转录cDNA

C. cDNA synthesis from total ribonucleic acid(TNA) or total RNA containing trace amount of genomic DNA

1). Add into 0.6 ml tubes: 12μI liquid wax, 2μl TNA (200-400 ng/μl) and 1μl DNase I mixture, and incubate at 37 C for 30 min. After DNA digestion, heat samples at 75 C for 10 min to kill DNase 1 and chill on ice for 2 min.

DNase Master mix: One Sample DNase I(2U/μl)* 0.1μl

10 x # 2 restriction buffer (Gibco) 0.05μl

RNase inhibitor 0.05μl

H20 0.8μl

*If your DNase I is suppiied in high concentration, dilute DNase I with 40% glycerol and Ix #1 restriction buffer (Gibco) (final) to 2 U/μl. Too much DNase I may destroy your mRNA.

2). Add 3.00μI cDNA mixture, melt Iiquid wax in 37 C water bath. Mix and spin samples, and set on PCR machine for cDNA synthesis.

cDNA Master mix: One sample Final Conc (μl/μl)

5 x BRL Buffer 1.0μl 2/10

0.1 M DTT 0.50μl 1/10

Hexamer (50 uM) 0.2μl 0.4/10

(T)16 (50μM) 0.1 μl 0.2/10

4 dNTP (2.

5 mM) 1.0μl 2/10

RNase inhibitor 0.1 μl 0.5/1 0

Rev Trans (lnvitrogen, 200UIμ1) 0.1 μl 0.2/10

3). Mix and spin samples, and set on PCR for cDNA synthesis using the same program. Add 45μI H20 into the cDNA tube (1: 10 fold dilution), mix and spin for PCR amplification.

Note:

1. RT-PCR can be done by synthesizing cDNA using gene-specific 3' primer instead of hexamer and (T)16

2. This DNA digestion procedure can also be used for total RNA to get rid of DNA contamination. Majority of total RNA extraction methods have DNA contamination. Unless pCR primer set is designed to across intron, we use this method for all cDNA synthesis.

3.Total nucleic acid (TNA) can be used for cDNA synthesis without DNA digestion. In this case, PCR primers that across intron will amplify genomic DNA and cDNA at the same time. Genomic DNA products include intron fragment. We can quantitate mRNA against genomic DNA instead of housekeeping gene, like b-actin.