SMARTer_RACE_5_3__Kit_User_Manual_022714(12)

clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual

3. SMARTer® RACE 5’/3’ Kit User Manual RNA Purity

The purity of RNA is the key factor for successful cDNA synthesis and SMARTer RACE. The

presence of residual organics, metal ions, salt or nucleases in your RNA sample can have a large

impact on downstream enzymatic applications by inhibiting enzymatic activity or degrading the

RNA. We strongly recommend checking the stability of your RNA to ensure that it is free of

contaminants. Impurities such as salt or organic contaminants can be removed by repeated

ethanol precipitation, subsequent washing with 80% ethanol and the complete removal of all

remaining ethanol.

Since RNA stability is a good indicator of RNA purity, we strongly recommend checking the

stability of your RNA to ensure that it is free of contaminants.

Incubate a small portion of your RNA at 37°C for 2 hours, then compare the sample to a duplicate

control stored at –70°C. If the sample incubated at 37°C shows a lower 28S:18S ratio than the

control or a significant downward shift on a formaldehyde agarose gel, the RNA may have

nuclease contaminants (see Section V.C., below, for methods for assessing RNA quality).

If your RNA template is from a plant or some other species with high pigment levels, please

pay special attention to polysaccharide/pigment contamination. Polysaccharides/pigments are

hard to remove and can’t be detected on the agarose gel. These glycoproteins might interfere with

primer binding sites of RNA during the first-strand cDNA synthesis leading to reduced cDNA

yield.

C. Assessing RNA Template Quality

1. Methods for Assessing Total RNA Integrity

Detection with the Agilent 2100 BioAnalyzer (Agilent Technologies, CA):

This microfluidics-based technology, which provides an alternative to traditional gel-

based analysis, requires only 2–7 ng of RNA per analysis. We recommend using RNA

samples with an RNA Integrity Number (RIN) of 7 or higher. In addition to assessing

RNA quality, this automated system provides a good estimate of RNA concentration.

If you do not have access to an Agilent 2100 BioAnalyzer, you can visualize your RNA

on a denaturing formaldehyde agarose gel under UV light. The theoretical 28S:18S ratio

for eukaryotic RNA is approximately 2:1. If the 28S:18S ratio of your RNA is less than 1,

your RNA template is not suitable for SMARTer RACE. When visualizing RNA using

EtBr, you need at least 0.5–1 µg of total RNA. Alternatively, SYBR® Green II or SYBR

Gold dyes (Molecular Probes; Eugene OR), allow you to detect as little as 1 or 2 ng of

RNA on your gel, respectively.

Methods for Assessing mRNA Integrity

All of the methods mentioned above can be used to assess the quality of your mRNA. However,

because mRNA does not contain strong ribosomal bands, the assessment of its quality will be

somewhat subjective. Typically, mRNA appears as a smear between 0.5 kb to 6 kb, with an area

of higher intensity around 1.5 and 2 kb. This size distribution may be tissue or species-specific. If

the average size of your mRNA is less than 1.5 kb, it could be an indication of degradation. 2.

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