ABI PRISM 7000 Sequence Detection Systems Relative ...

5′ 3′
5′ cDNA
Reverse
Primer
Oligo d(T) or random hexamer
3′ 5′
cDNA
Generating cDNA
Storing cDNA
Primer Extended on mRNA
Synthesis of 1st cDNA strand
Storing cDNA
After thermal cycling, store all cDNA samples at −15 to −25 ° C. To minimize repeated
freeze-thaw cycles of cDNA, Applied Biosystems recommends that you store your
cDNA samples in aliquots.
CHEMICAL HAZARD. 10 × RT Buffer may cause eye, skin, and
respiratory tract irritation. Read the MSDS, and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves.
Example
For the sample experiment, RNA was extracted from the liver, bladder, and kidney tissues of an individual. RNA
concentration was determined through spectrophotometry (using A 260 ) and the RNA was diluted to a final concentration of
50 ng/µL.
The RT master mix was prepared as follows, using guidelines from the High Capacity cDNA Archive Kit Protocol:
Component
Volume (µL)/Reaction
10✕ Reverse Transcription Buffer 10
25✕ dNTPs 4
10✕ random primers 10
MultiScribe Reverse Transcriptase, 50 U/µL 5
Nuclease-free water 21
Total per reaction 50
The cDNA archive plate was then prepared by pipetting
• 50 µL of the RT master mix
• 30 µL of nuclease-free water
• 20 µL of RNA sample (bringing the total starting amount of RNA to 1 µg per 100 µL reaction)
The RNA was then converted to cDNA using the universal thermal cycling parameters for two-step RT-PCR, as described in
“Thermal Cycling Parameters for RT” on page 16.
The cDNA was stored at −20 ° C for 24 hours.
3
DRAFT
September 26, 2003 3:14 pm, C3_RT.fm
Notes
Relative Quantification Getting Started Guide for 7000 v1.1 17