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 >8
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 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.
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’ adapter ligated
small RNA fragments
5’ & 3’ adapter ligated
small RNA fragments

5. 6. Reverse Transcribe and Amplify the Small RNA Ligated with Adapters
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. 3’ 5’ 5’ 3’
Purified 5’ and 3’ ligated RNA
Reverse Transcription with
SuperScript III Reverse Transcriptase –
First strand synthesis generating single
Strand cDNA 3’ 5’
cDNA strand 5’ 3’
RNA strand
RNase OUT denatures RNA strand 3’ 5’
PCR amplification generates second
cDNA strand and enriches adapter ligated
products 3’ 5’ 5’ 3’

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 ng 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 ng of DNA.
Measure the OD 260/280 ratio, which should be ~1.8.
Sample
Ladder
Cut from gel
and purify