Small RNA Sample Preparation

1
Small RNA Sample Preparation
1. Quality check of total RNA
Customers should carry out a quality check
of their total RNA by running it out on a 1
agarose gel, and the integrity of RNA judged upon
staining with ethidium bromide. High quality,
intact RNA will show a 28S rRNA band at 4.5kb,
that should be about twice the intensity of
the 18S rRNA band at 1.9kb. Both kb
determinations are relative to a 1kb ladder. The
mRNA will appear as A smear from
0.5-6kb. Completely degraded RNA will appear as a
very low molecular weight smear.
Customers are to supply 10ug of purified total RNA
2
Small RNA Sample Preparation
2. RNA Quality Check by Agilent 2100 Bioanalyzer
18S rRNA
28S rRNA
Marker
The AWCGS runs an isolated RNA quality check by
Agilent 2100 Bioanalyzer system. The Bioanalyzer
generates an RNA integrity number (RIN). RNA
samples with RIN gt8 Will be further processed for
Solexa expression analysis.
3
Small RNA Sample Preparation
3. Isolate Small RNA by Denaturing PAGE Gel
100bp
80bp
60bp
40bp
20bp
10ug of total RNA is run on a 15 TBE-urea
gel for 1 hour at 200V with a size ladder in 20bp
steps Up to 100bp.
Cut out a band of gel corresponding to the 18-30
nucleotide bands in the marker lane, and gel
extract.
4
Small RNA Sample Preparation
4. Ligate 5 Adapter
5 small RNA adapters
A single stranded 5 small RNA adapter is
ligated to the 5 end of each small RNA
fragment. The 5 small RNA adapter is necessary
for reverse transcription and amplification of
the small RNA fragment. This adapter also
contains the DNA sequencing primer binding site.
Gel purify the adapter modified small
RNA Products on a 15 TBE-urea gel, and
excise And purify the products in the 40-60nt
size range.
Small RNA fragments 18-30nt in length
3 small RNA adapters
5. Ligate 3 Adapter
A single stranded 3 small RNA adapter is
ligated to the 3 end of each small RNA fragment.
The 3 Small RNA adapter corresponds to the
surface bound amplification primer on the flow
cell used for Cluster generation.
5 adapter ligated small RNA fragments
5 3 adapter ligated small RNA fragments
Gel purify the adapter modified small
RNA Products on a 10 TBE-urea gel, and
excise And purify the products in the 70-90nt
size range.
5
Small RNA Sample Preparation
6. Reverse Transcribe and Amplify the Small RNA
Ligated with Adapters
Reverse transcription followed by PCR is used to
create cDNA constructs based on the small RNA
ligated with 5 and 3 adapters. This protocol
selectively enriches those RNA fragments that
have adapter molecules on both ends. The PCR is
performed with two primers that anneal the ends
of the adapters.
5
3
5
3
Purified 5 and 3 ligated RNA
Reverse Transcription with SuperScript III
Reverse Transcriptase First strand synthesis
generating single Strand cDNA
5
cDNA strand
3
3
5
RNA strand
RNase OUT denatures RNA strand
5
3
PCR amplification generates second cDNA strand
and enriches adapter ligated products
5
3
3
5
6
Small RNA Sample Preparation
7. Purify the Amplified cDNA Construct
Gel purify the adapter modified cDNA construct on
a 6 TBE PAGE gel, and excise and purify the
products in the 92bp size range.
25bp ladder is in 25bp Steps up to 300bp
100bp
8. Validate the Library
92bp
75bp
Determine the molar concentration of the library
ready for Cluster Generation
50bp
25bp
Run the products on an Agilent 2100 Bioanalyzer,
to check The size, purity, and concentration of
the sample. The yield should be 500-100ng of
DNA. Measure the OD 260/280 ratio, which should
be 1.8.
Run the products on an Agilent 2100 Bioanalyzer,
to check The size, purity, and concentration of
the sample. The yield should be 500-100ng of
DNA. Measure the OD 260/280 ratio, which should
be 1.8.
Sample
Ladder
Cut from gel and purify