Biomedical Engineering Reference
In-Depth Information
Fig. 2. RNA quality check before starting RT reactions. These are striatal RNA
samples from eight different rats, extracted using TriReagent and treated with DNase I.
The 28S and 18S ribosomal bands should be clearly visible, the background mRNA
smear should be of similar size to the rRNA bands, and there should be little or no
lane-to-lane variation.
2. For each RNA sample, there will be three RT reactions—one for each of the three
anchored 3
primers (“A,” “C,” “G”). In each 0.2-mL tube, add the appropriate RT
primer (2
L
of RT mix, vortex-mix, touch-spin, and immediately place into a preheated
(65°C) thermal cycler. Start following program: 65°C for 5 min, 37°C for 60
min, 95°C for 5 min,
µ
L of 2
µ
M
), 0.2
µ
g of RNA, and H
2
O to 4
µ
L total. Then add 15
µ
4°C. After 10 min of the 37°C period, carefully add 1
µ
L of the RT enzyme directly into each reaction, then rapidly pipet up and down
a few times to mix (
see
Note 7
). RT products are best used immediately, but
can also be frozen (at -20°C) and reused for several weeks. Depending on your
experimental design, there will generally be enough cDNA for four or more
sets of PCR reactions.
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