Small RNA Extraction using Fractionation Approach and
Library Preparation for NGS Platform
Spandan Chaudhary1*, Pooja S. Chaudhary2 and Toral A. Vaishnani3
1Department of Medical Genetics, Xcelris Labs Limited, Old Premchandnagar Road, Opp. Satyagrah Chhavani, Bodakdev, Ahmedabad-380015, Gujarat, India
2NGS department, Xcelris Labs Limited, Old Premchandnagar Road, Opp. Satyagrah Chhavani, Bodakdev, Ahmedabad-380015, Gujarat, India
3Bioinformatics department, Xcelris Labs Limited, Old Premchandnagar Road, Opp. Satyagrah Chhavani, Bodakdev, Ahmedabad-380015, Gujarat, India
Dr. Spandan Chaudhary, Department of Medical Genetics, Xcelris Labs Limited, Old Premchandnagar Road, Opp. Satyagrah
Chhavani, Bodakdev, Ahmedabad-380015, Gujarat, India, E-mail:
Received: March 15, 2016; Accepted: April 04, 2016; Published: April 12, 2016
Small RNA isolation is a herculean task; it requires lots of
standardization at each and every step. In present study, we have
combined two protocols of total RNA isolation and standardized
the protocol for small RNA isolation using flash page fractionation
method. Due to difficulty in isolating high quality small RNA, most
technologies use total RNA as a starting material for preparing small
RNA library. Quality of both total and small RNA was determined
using scientifically proven technology like Agilent bioanalyzer. In
present study we have demonstrated a protocol for total and small
RNA isolation from rice, followed by small RNA library preparation
using Solid total RNA seq kit protocol for NGS platform (For SOLiD
platform). Small RNA library was of good quality as per the parameters
given in the protocol. We have validated the procedure four times
which resulted good data, it concludes these procedures can be use to
isolate high quality small RNA to be used for deep sequencing.
Keywords: miRNA; Small RNA; Flash page fractionators; Small
RNA library; NGS; SOLiD
Small RNA pairs with their target messenger RNA molecules
and to suppress the gene expression in order to control the gene
expression. miRNAs play a major role in cell proliferation, cell
cycle, cell differentiation, metabolism, apoptosis, developmental
timing, neuronal cell fate, neuronal gene expression, brain
morphogenesis, muscle differentiation and stem cell division. The
miRNAs are small, highly conserved RNA molecules that act as
key regulators of development, cell proliferation, differentiation,
and the cell cycle. Emerging evidence also implicates miRNAs in
the pathogenesis of human diseases such as cancers, metabolic
diseases, neurological disorders, infectious diseases and other
illnesses [1-2]. Small RNAs have been classified into at least six
groups, which are microRNAs (miRNAs), heterochromatic small
interfering RNAs (hc-siRNAs), trans -acting small interfering
RNAs (tasiRNAs), natural antisense small interfering RNAs
(nat-siRNAs), repeat-associated small interfering RNAs (rasiRNAs),
and in metazoans, the piwi-interacting RNAs (piRNAs)
[3-7]. The active mature miRNAs are typically 21-24 nucleotides, single stranded RNA molecules expressed in eukaryotic cells.
Small RNAs are 21-24 nucleotides in length and are known to
play a major role in the activation of mRNAs and genomic DNAs
. 21-nucleotide microRNAs (miRNAs) and 24-nucleotide
Pol IV-dependent small interfering RNAs (p4-siRNAs) are the
most abundant types of small RNAs in angiosperms . Some
miRNAs are well conserved among different plant lineages;
whereas others are less conserved, and it is not clear whether
less-conserved miRNAs have the same functionality as the well
conserved ones . Recent whole genome sequencing data
indicates that the 5’- and 3’- ends of miRNAs are variable, in
which 5´end is less variable than 3´ end . These alternative
length miRNAs are called isomers, and their biological function
is unknown, miRNAs are known to affect the translation and/
or stability of the target messenger RNAs . Each miRNA is
believed to regulate multiple genes, and it is currently thought
that greater than one third of all human genes may be regulated
by miRNA molecules.
Small RNA was first discovered by David’s Baulcombe's
group at the Samsbury laboratory in Norwich, England, as a part
of post transcriptional gene silencing in plants. RNA interference
invariably leads to gene silencing via remodeling chromatin
to thereby suppress transcription, degrading complementary
miRNA or blocking protein translation . Small RNAs are
naturally produced as part of the RNA interference (RNAi)
pathway by the enzyme dicer. SiRNAs are short double stranded
RNA with 2 nucleotide overhangs on either end including a 5'-phosphate group and a 3'- hydroxyl(-OH) group .
Rice is a staple food in India and a very important part
of appetite of the entire world. It is one of the most important
cereal and model monocot plant [14,15]. About three billion
of the population depends on rice for their daily calorie needs
. Plenty of research work is going on and rice genome is
also available, so it’s comparatively easy to identify and study
the siRNAs. There are two species: Oryza sativa and Oryza
glaberrima, first being the most commonly grown throughout
Asia, Australia, the Americas and Africa; and second species is
grown on a small scale in western Africa . In present study, we have standardized the protocol for small RNA extraction from
4 rice samples, suitable for downstream application like small
RNA sequencing using next generation sequencing technologies
like Miseq, NextSeq, HiSeq, Solid, Ion PGM etc. But, we have
demonstrated the standardized protocol of preparing libraries
for Solid analyzer. In any of the next generation platform for
getting the best data from small RNA sequencing, quality of small
RNA is very crucial criteria. Because of the complexity of the small
RNA isolation protocols, Illumina is referring poly acryl amide
gel based method to extract small RNA from adapter ligated total
Small RNA can be isolated from any tissue or plants directly
using commercially available kits like mirVana™ miRNA Isolation
Kit (Thermo scientific), miRNeasy Mini Kit (Qiagen) etc uses dual
column based method in which small RNA is bound to second
column. There is also a kit available which directly captures small
RNA from tissue like mirPremier microRNA Isolation Kit (Sigma
Aldrich). Common chemistry shared by all this kit is pH based
binding of nucleic acid to the silica column and these columns
are made to bind certain specific size of nucleic acids. On the
other hand, flash PAGE Fractionator works on the principle of gel
electrophoresis which isolates the molecules on the bases of the
size which helps in efficient small RNA extraction. Column based
methods isolate small RNA upto 200 nucleotides which captures
data in deep sequencing technologies where as small RNAs like
piRNA or siRNA are of 21-24 nucleotides long which are the
desired target for any study, can be isolated precisely using flash
Materials and Methods
Prior to the harvesting, juvenile leaves of rice were washed
with DEPC (Diethylpyrocarbonate- D5758, Sigma ) treated
water to remove surface contamination at sample collection site
Jetalpur, Ahmedabad (Gujarat) India and were transported in
TMS RNA stabilizer solution (XGgtms-100) with dry ice followed
by storage at -80°C.
Total RNA isolation
Rice leaves were thawed at room temperature 100 mg and
and taken for RNA isolation using pure Link miRNA isolation
kit (Invitrogen- cat. no. K1570-01) and pure Link RNA Micro kit
(Invitrogen- cat. no. 12183-016) as per manufacturer’s protocol.
Further, combination of both these kit were used to get high
quality and yield of total RNA. Total RNA isolation was done in
triplicate using three different methods. Combination of both
these kit's methods are as follow:
100 mg of plant leaves were grounded to fine powder in
motor and pestle with liquid nitrogen. 300 μl of binding buffer
(L3) (supplied with miRNA isolation kit) was added to powder in
mortar pestle and ground it gently to mix properly till it become
complete homogenous solution. Lysate was centrifuged at 12000
g for 2 minutes at room temperature to remove any particular
material. Supernatant was transferred to another sterile micro
centrifuge tube and 300 μl of 70% of ethanol was added to lysate and vortexed to mix well. Whole lysate volume 600 μl was loaded
on a new spin cartridge and centrifuged it at 12000 g for 1 minute
at room temperature. 350 μl of Wash Buffer I was added to spin
cartridge and centrifuged at 12000 g for 15 seconds at room
temperature and flow through was discarded. In a separate 0.2 ml
tube 70 μl of DNase buffer and 20 μl of DNase-I enzyme (Thermo
scientific cat. no. ENO525) was taken (Total 80 μl) and mixed by
pipetting and kept on ice. DNase I mixture (80 μl) was added to
the center of the cartridge and kept on ice for 15 minutes. After
15 minutes again 350 μl of Wash Buffer I was added to the center
of the spin cartridge and centrifuged at 12000 g for 15 seconds
at room temperature and flow through was discarded. Spin
cartridge was transferred to a clean RNA wash tube, provided
with the kit and 500 μl of Wash Buffer II with ethanol was added.
Spin cartridge was centrifuged at 12000 g for 15 seconds at room
temperature and flow through was discarded. Above two steps
were repeated and spin cartridge was centrifuged at 12000 g for
1 minute at room temperature to dry the membrane. Collection
tube was discarded and cartridge was inserted into RNA recovery
tube supplied with the kit. 30 μl of RNase- free water was added
to the center of spin cartridge and incubated at room temperature
for 1 minute then centrifuged for 2 minute at ≥ 12000 g at room
temperature to elute the total RNA. Above step was repeated
with 30 μl of RNase- free water with same collection tube.
Quantitative and qualitative determination of RNA
Concentration and purity of total RNA samples were
measured using the Nano Drop ND3.0 spectrophotometer
(NanoDrop Technologies Inc, Wilmington, DE). For preparing
1% denaturing agarose gel, 0.5 g of agar powder was added to
50 ml of DEPC treated water and boiled till it melted, followed by
adding 8.75 ml of formaldehyde and 5 ml of 10X mops. Gel was
run at 90Volts for about 45 minutes (Figure 1). RNA integrity was
assessed using nano chip on Agilent 2100 Bio analyzer (Agilent
Technologies, Palo Alto, CA) (Figure 2). The gel image and bio
analyzer profile with RIN value were analyzed to proceed with
small RNA enrichment step.
Small RNA enrichment
Flash page fractionator (Ambion cat no.13100) (Figure 3) is a
miniaturized version of poly acryl amide gel electrophoresis for
isolating small RNA from total RNA samples. For the enrichment
of small RNA using flash page fractionator system, four other
materials are required.
1. Flash page Pre-Cast gel (Type A) (Cat no. 10010- ambion)
2. Flash page buffer kit (Type A) (Cat no. 9015- ambion)
3. ElectroZap™ Electrode Decontamination Solution (Cat
4. Flash page Reaction Clean-up Kit (cat no.Am12200).
60μg of high quality (RIN value above 6) total RNA was taken
with loading buffer and dye, heat denatured and loaded onto the
upper gel surface in the flash page fractionator, electrophoresed
at 80V constant voltage until the blue dye begins to exit the gel.
As per manufacture´s protocol this time should be around 12
Figure 1: Agarose denaturing gel profile of total RNA isolated by combined pureLink miRNA isolation kit (invitrogen) and pureLink RNA Micro kit.
Sample-1, 2, 3, 4 were loaded in lane 1, 2, 3, 4 respectively.
Figure 2: Total RNA profiling of samples 1, 2, 3 and 4 as figures 2a, 2b, c and 2d respectively, run on Agilent bioanalyzer nano chip.
minutes to obtain small RNAs with length of 15-140 nucleotides
in the lower reservoir of the flash page system. As the selection
of the small RNA is very important aspect of small RNA library
preparation this step has also similar importance. The time
described in the manufacturer protocol is mainly depends upon
Figure 3: Flash page fractionator from Ambion (Thermo Fisher Scientific).
the amount and quality of total RNA used, we had standardized
the time for small RNA isolation is 13-14 minutes with the
supplied pre-cast gels. The time required to obtain small RNA in
the lower reservoir depend upon age of the pre-cast gel, as it is
near to the expiry it will take more time which can be ranging
from 12-15 minutes. Further, small RNAs were purified using
flash page reaction clean-up kit (Ambion) as per manufacturer's
protocol and small RNA profile was checked on Agilent 2100 bio
analyzer using small RNA chips (cat no.5067-1548) (Figure 4).
Handling of small RNA reagents for running on bio analyzer chip
is difficult because it uses highly concentrated gel to separate
small RNA fragments which are 21-24 nucleotides in length.
Usually bioanalyzer protocols recommend use of filtered gel
matrix within one month of time but it is not suitable with small
RNA gel matrix. If stored gel matrix is used it will not detect
the chip on bioanalyzer which was very usual case with small
RNA chips the reason behind this is electrode cannot sense the
gel matrix of the chip. To overcome this we used silica column
(Nanosep centrifugal Devices with Omega Membrane, cat.
no.29300-610) to prepare fresh gel matrix as agilent kit provides
Figure 4: Small RNA profiling of samples 1, 2, 3 and 4 as figures 4a, 4b, 4c and 4d respectively, run on Agilent bioanalyzer small RNA chip.
two vials of gel matrix 650 μl volume in each with two spin filters,
however quantity is enough to run 25 chips. For preparing fresh
gel matrix 45 μl of gel was filtered each time through the column.
Two micro liter dye (provided in the kit) was added to 40 μl of
filtered gel and rest protocol was followed as per manufacturer's
Small RNA cDNA library preparation
Small RNA cDNA library was prepared using Solid™ Total
RNA-Seq Kit (Part Number 4452437 Rev. A 01/2010)  which
involve five steps described as follows:
Hybridization and adapter ligation
The 5'- adapter ligation reaction was carried out in 20 μl
reaction containing 80-100 ng (as per nanodrop readings) of
enriched small RNA in 3 μl volume, 3 μl hybridization solution, 2
μl solid adapter mix, 8 μl of 2X ligation buffer and 2 μl of ligation
enzyme. Reaction mixture was incubated at 16 degree for 16 hrs
in a thermal cycler with heated lid open.
RT-PCR of adapter ligated small RNAs
After 16 hours of incubation, reverse transcription was
carried out using 20 μl reaction, containing 4 μl 10X RT buffer,
2 μl dNTP mixes, 2 μl solid RT primer and 11 μl nuclease free
water. The reaction mixture was heat denatured at 70 degree for
5 minutes followed by snap chilling then 1 μl array script reverse
transcriptase enzyme was added in the reaction mixture and was
incubated at 42 degree for 30 minutes resulting into the formation
of single stranded cDNA followed by purification by using
minelute PCR purification kit (Qiagen). The single stranded cDNA
was quantified by using nanodrop 8000 spectrophotometer.
Size selection for library preparation
10% TBE urea gel 1.0 mM, 10 wells (invitrogen cat no.EC68752BOX) was used for size selection. Single stranded
cDNA samples; 100ng of 10bp ladder (Cat no.10821015
invitrogen) at the concentration of 40 ng/ μl was loaded in the
gel. The gel was run in x cell sure lock mini cell gel assembly using
1X TBE running buffer at 180 volts for 47 minutes instead of 45
minutes recommended in the protocol. Gel was stained for 10
minutes with gentle agitation using syber gold nucleic acid gel
stain. A continuous smear was seen in the SS cDNA well (Figure
5). The cycles for the in gel amplification would be determined on
the basis of intensity of smear. Gel was cut horizontally between
60-80 nucleotides and gel slices was further cut vertically into 4
pieces of around 1mm X 6mm using clean blade. All four pieces
of the gel slices were transferred to fresh 0.2ml RNAs free tubes.
In Gel PCR amplification and purification
Out Of four gel slices, 2nd and 3rd gel slices were used first for
in gel PCR amplification. This 100 μl reaction contains 10 μl of
PCR buffer, 8 μl 2.5 mm dNTP, 2 μl solid 5' primer, 2 μl solid 3'
primer, 1.2 μl amplitaq DNA polymerase and 76.8 μl nuclease free
water and the gel slice as template DNA. 18 cycles of amplification
was carried out and checked on E-gel 2% size select gel (cat.
Quantification and qualification of cDNA library
This cDNA library was purified using pure link PCR micro kit
(Invitrogen) and further, quantified and qualified using 2100 bio
analyzer with agilent high sensitivity (HS) DNA kit (cat no. 5067-
4626) (Figure 6).
Result and Discussion
Ethanol precipitation method along with nucleic acid carrier
such as glycogen is recommended for extracting total RNA with
sizes of RNA having from ∼ 20 nt to several kilo bases in length.
For RNA isolation, either reagent based or silica membrane
Figure 5: Gel image of size selection using10% TBE urea gel 1.0 mM, 10 wells invitrogen cat no. EC68752 along with 10bp Invitrogen ladder in well
labeled as M, samples are labeled as S1, S2, S3 and S4.
Figure 6: Small RNA library profile of samples 1, 2, 3 and 4 as figures 6a, 6b, 6c and 6d respectively, run using high sensitivity DNA chip on Agilent
based methods are generally used but it was found that small
RNAs cannot be isolated using silica column based method
instead trizol reagent based method is more suitable for the
same , but we extracted total RNA using a modified silica
column based method and isolated high quality total RNA with
RIN value above 6, from this total RNA samples small RNA was
isolated and checked on Agilent 2100 bioanalyzer. (Agilent
Technologies, Santa Clara, Palo Alto, CA, USA). For getting good
data by deep sequencing the most important parameter is the
good quality library preparation which depends on the quality
of total RNA and small RNA. RNA degradation during isolation
procedure or loss of small RNA is the main reason for failure to
obtain a good library. Absorbance 260/280 ratio was found to be
1.9, with the total yield of 90 micrograms, total RNA was loaded
on 1% denaturing agarose gel, 2 distinct bands were observed,
first band was of 28s rRNA and second band was of 18s rRNA
(Figure 1). In Bio-analyzer profile, distinct peaks of 28s and 18s
rRNA were visible along with a small peak near the lower marker indicating 5s rRNA region.
The quality of total RNA and selection of small RNA
enrichment procedure play a momentous role in the small RNA
cDNA library profiling. Total RNA having RIN value below 6
normally could not be used for small RNA enrichment, because
such RNAs are found to be degraded. Several kits are available
in the market that isolate small RNA directly from the tissues
which don’t require total RNA isolation to be done separately
like Invitrogen miRNA isolation kit. But a drawback of this small
RNA isolation method is that along with small RNA mRNAs
and some other RNAs get also isolated which hinders in library
preparation process and grab the data in the deep sequencing
process. Flash page fractionators works as a vertical gel unit, it
has negative electrode on the top and positive electrode at the
bottom, RNA will migrate from upper side to the lower side. Same
like normal gel electrophoresis principle, smaller fragments will
migrate faster than the longer ones. When total RNA degradation
occurs, 28s ribosomal RNA 18s ribosomal RNA band degrades
Figure 7: Orientation of RNA sequences, adapter sequence and barcode for Solid System sequencing
Table 1: Input amount of starting material and amount of resulted product.
End Product (Average)
Total RNA isolation
100 mg leaves
60 µg total RNA
Small RNA enrichment
60 µg total RNA
350 ng small RNA
Hybridization and adapter ligation
100 ng of enriched small RNA
155 ng single stranded cDNA
and produces small size ribosomal RNA fragments  which
comes out with small RNA when pass through flash gel which
ultimately leads to failure of the library or contamination of small
RNA library with ribosomal reads. In order to avoid this, intact
total RNA without any degradation is advised to use for preparing
small RNA libraries even with flash page fractionators system.
NGS library preparation process requires optimization at
each and every step from nucleic acid isolation to library quality
check. Here, we have tried to provide the accurate data for how
much starting material is required at each step and how much
end product will be generated which is the principle missing
point in all the available protocols, these will help the readers to
repeat the same experiment. For small RNA enrichment by flash
page fractionators, up to 100 μg of high quality total RNA with
RIN value of above 6 is advice. In present study, we started with
60 μg of high quality total RNA having RIN value 6.2 to 6.8, and
after purification we obtained 350 ng of enriched small RNA.
Small RNA profiles were analyzed using Agilent small RNA chip
(shown in figure 4.) and the amount of small RNA was calculated
and 100 ng small RNA was taken for adapter ligation.
The RNA samples are hybridized and ligated with the
adapters containing set of oligonucleotides with a single-stranded
degenerate sequence at one end and defined sequence required
sequencing at the other end. The adapters ligate in a manner that
it provides template for sequencing from the 5′ end of the sense
strand. The downstream emulsion PCR primer alignment and the
resulting products of template bead preparation for Solid System
sequencing are illustrated in (figure 7) Accurate size selection is
very important for any library preparation for deep sequencing.
Size selection was done using 10 bp ladders as a reference; the
gel was cut between 60-80 nucleotides. The insert length of the
adapters is 18-38 bp, so the length of the amplified PCR product
will be 110-130 bp but ideally small RNA library bands obtained
within the range of 120-130 bp.
According to the protocol Solid TM Total RNA-Seq kit,
criteria for small RNA cDNA library to be used for sequencing run depends upon the values obtained from the smear analysis done
on DNA 1000 chip run. In present study we have used DNA High
sensitivity chip so we modified the smear analysis criteria also.
As per protocol, the library will be used for sequencing only if
the ratio of 120-130 bp area / 25-150 bp area is more than 50%,
if this ratio is less than 50%, second round of size selection will
be required. But as we have used high sensitivity chip, in which
lower marker peak is at 35 bp instead of 15 bp in DNA 1000 chip,
we have calculated the ratio 40-150 bp area. High sensitivity chip
was used instead of DNA 1000 chip. In case of plants, it is very
difficult to obtain small RNA libraries with >50% ratio but in
the present study we have achieved 52% ratio in one of our four
Total RNA isolation protocol prepared by combining two
different kits worked efficiently and provided high quality
total RNA. Small RNA isolated from total RNA using flash page
fractionator was of the good quality to be used for preparing small
RNA libraries for all the NGS platforms. The libraries prepared
by modified method were used for sequencing using Solid
analyzer and generated good quality data (data not provided)
which proves the modification applied at each stage of library
preparation are acceptable.
Authors' are thankful to the management of Xcelris Labs for
providing financial support.
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