Biology Reference
In-Depth Information
Bioanalyzer. This method replaces the traditional running of
the sample through an agarose gel, which is an acceptable
alternative if this instrument is not available.
3. RNA samples are always kept on ice or stored at −80°C.
Samples selected for TaqMan miRNA array studies should
meet the following quality requirements:
(a) A 260/ A 280 ratio should be ³ 2.0.
(b) A 260/ A 230 ratio should be ³1.8.
(c) 28S/18S rRNA ratio should be ³1.5.
(d) RIN should be ³8.0.
(e) The method used to isolate the total RNA must preserve
the small RNA fraction. However, in order to preserve endog-
enous control sequences present in the total RNA (longer
fraction), do not enrich for the small RNA fraction. This
may cause the control transcripts to be lost. Isolation of a
clean total RNA fraction allows other applications to be
performed on the exact same RNA sample, such as exami-
nation of mRNA expression.
4. These methods of RNA detection are described in the manu-
facturer's protocol.
3.4. Stem-Loop
Reverse Transcription
Reaction
1. Real-time PCR is recommended to be used in combination
with stem-loop RT to quantify miRNA, since stem-loop RT
primers are better than conventional ones in terms of RT
effi ciency and specifi city ( 5 ).
2. Each RT reaction contains:
(a) 3 ml RNA sample (1-1,000 ng) plus nuclease-free water.
(b) 4.5 ml RT reaction mix containing:
Megaplex RT Primer, Rodent Pool A:
-
0.8
ml Megaplex RT Primers (10×).
0.9
- ml MgCl 2 (25 mM).
TaqMan MicroRNA RT Kit:
-
0.2
ml dNTPs with dTTP (100 mM).
-
1.5
ml MultiScribe reverse transcriptase (50 U/ml).
0.8
-
ml RT buffer (10×).
0.1
-
ml RNase inhibitor (20 U/ml).
0.2
- ml nuclease-free water.
The reaction mix can be prepared in bulk for all reactions
3. The 7.5 ml reactions are incubated in the GeneAmp 9700
Thermocycler in either a 96-well plate or 8-tube strips for
and then dispensed (4.5 ml) into each reaction tube.
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