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Old 09-24-2013, 06:07 AM   #1
frang11
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Default Small RNA sequencing problem

Hello everybody.
We have a problem with Illumina sequencing of miRNA contained in mouse serum. It is the first time we prepare a small RNA library, thus we are not able to understand where is our problem since we followed the protocol step by step. The blood was taken by cardiac puncture on anaesthetized mice.We have then isolated small RNAs using de miRNAeasy serum/plasma kit (QIAGEN) and made a library for Illumina sequencing using NEbNext® Multiplex Small RNaLibrary Prep Set for Illumina® (NEB). Before start the protocol we analyze the RNAs profiles by Nanodrop, the result were not the best, but since many articles and also the Illumina support, suggest to work in volumes because is not possible quantify exactly the smallRNA fraction, we pooled 3 serum samples of three different treatments. Each pool was made by equal volumes of serum and we assigned a different Index of the NEbNext® kit for each pool. After PCR we made a size selection with AMPure XP beads. The bioanalyzer profile showed many little peaks with a major band at 151 and 154 bp depending on the sample. We have sequenced 36 pb and found almost no miRNA. Instead, almost all the reads we have are for tRNA. Does anyone have had this problem before? Could it be the size selection with the beas was not so efficient? Is it a technical problem or it came from the bioinformatic analysis? All suggestions to resolve our problem are welcome!!
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Old 09-24-2013, 01:03 PM   #2
kcchan
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Small RNA libraries should appear as a band between 141-146bp. AMPure bead purification is not recommended for small RNA library generation. The purification is too imprecise to purify such small PCR products. Running a gel is still the best way to go.
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Old 09-24-2013, 01:50 PM   #3
frang11
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Thanks a lot Kcchan. The gel purification was our idea at the beginning but at the senquencing service of the University of Laval, they told us was unnecessary. I will reset my protocol as soon as possible to have better result. Do you have any idea why in our samples we have a shift in the main band? why the main peak is at 153? Could it be an effect of the low quality samples?
Sorry for asking a lot of question but we would really like figure out every details
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Old 09-24-2013, 01:52 PM   #4
frang11
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Thanks a lot Kcchan. The gel purification was our idea at the beginning but at the senquencing service of the University of Laval, they told us was unnecessary. I will reset my protocol as soon as possible to have better result. Do you have any idea why in our samples we have a shift in the main band? why the main peak is at 153? Could it be an effect of the low quality samples?
Sorry for asking a lot of question but we would really like figure out every details

Last edited by frang11; 09-24-2013 at 01:54 PM. Reason: posted twice
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Old 11-09-2013, 04:19 AM   #5
victorsor
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Quote:
Originally Posted by frang11 View Post
Thanks a lot Kcchan. The gel purification was our idea at the beginning but at the senquencing service of the University of Laval, they told us was unnecessary. I will reset my protocol as soon as possible to have better result. Do you have any idea why in our samples we have a shift in the main band? why the main peak is at 153? Could it be an effect of the low quality samples?
Sorry for asking a lot of question but we would really like figure out every details
I agree with Mcchan in that the best method for size selection is the gel cut. We have obtained very good results both with Truseq and NEBnext. However the size that you can be obtained depend of nature of sample (about 145 pb miRNAs and 155 pb piwi RNAs). I suggest you see the TruSeqSmallRNA Sample PreparationGuide pages 27-28. In attachment you can see our last library prepared with NEBnext.
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Old 11-11-2013, 06:12 AM   #6
Martin Mikkelsen
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Does anyone have experience using a LabChip XT for this?

We are thinking of using one, but we need to figure out the correct setting. We have tried the application note from PerkinElmer but we have problems using the automated peak recognition.
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Old 04-24-2014, 01:36 AM   #7
erlandsen
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Quote:
Originally Posted by Martin Mikkelsen View Post
Does anyone have experience using a LabChip XT for this?

We are thinking of using one, but we need to figure out the correct setting. We have tried the application note from PerkinElmer but we have problems using the automated peak recognition.
Hi Martin Mikkelsen.
I also tried PE's technote how to do size selection on the Labchip XT, and it did not work for us either. I ended up with tuning inn an area 140 bp +/- 8%, bye doing that I had to assume all libraries would behave the same, and I also had to run a ladder on each chip. But it looks as if it has worked well, average size of 96 samples is 143 bp.

Regards,

erlandsen
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Old 04-25-2014, 12:33 AM   #8
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Regarding the gel purification: What kind of filter do you use? E.g. Illumina recommends 5 µm filter tubes by IST Engineering. Millipore also offers 5 µm spin filters. And then there is the classic way of using silanized glass wool.
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Old 06-27-2014, 03:30 AM   #9
Petrinni
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Quote:
Originally Posted by victorsor View Post
I agree with Mcchan in that the best method for size selection is the gel cut. We have obtained very good results both with Truseq and NEBnext. However the size that you can be obtained depend of nature of sample (about 145 pb miRNAs and 155 pb piwi RNAs). I suggest you see the TruSeqSmallRNA Sample PreparationGuide pages 27-28. In attachment you can see our last library prepared with NEBnext.
Hi Victorsor, I would like to ask you what is the difference between TruSeq and NEBnext for preparing the library for small RNA sequencing on MiSeq. We are now using TruSeq, but we have a problem with ligation of adapters. It seams to us that they are not ligated properly. We are using 1000ng of total RNA from serum samples, so maybe higher amount of RNA could work better? Do you have any advice how to improve adapter ligation? Thanks a lot.
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Old 06-30-2014, 07:11 AM   #10
huguesparri
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Quote:
Regarding the gel purification: What kind of filter do you use? E.g. Illumina recommends 5 µm filter tubes by IST Engineering. Millipore also offers 5 µm spin filters. And then there is the classic way of using silanized glass wool.
We used Spin-X from Costar and they worked fine.
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Old 08-22-2014, 04:26 AM   #11
AMumtaz
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Hi, I used the NEBNext small RNA multiplex kit as well and about 70% or my reads were tRNA. This was rRNA depleted sample, size selected (~155bp) by PAGE.
Very strange. Did you figure out why you had so many tRNA reads?.

Last edited by AMumtaz; 08-27-2014 at 08:50 AM.
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Old 11-19-2014, 01:29 AM   #12
mikaelk
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Hi,

I would recommend to gel purify your samples before starting the library construction AND after the PCR. I used for the first gel a ladder called ZR small RNA ladder from Zymo to correctly evaluate the size of miRNAs as for example the Illumina ladder were having a shift of 10 bases in my hands.

It is indeed longer but by doing so, I have been able to get really clean and good libraries.

I have been constructing libraries for some years now and one thing that is always true is the classical GIGO, garbage in --> garbage out. Either you filter directly when you construct the library or you sequence garbage that you have to filter bioinformatically.

The differences in size between the different small RNA populations is so small that you will always get contamination from other fractions but the gels really decrease the problem.

The size selection should be rather around 140 bp as indicated before. Adpater dimer being around 120bp, if you size select at 155bp, it means you have and insert size of 35bp that do not correspond to miRNAs...

In the best case scenario, one should size select first between 18 and 30nt on 15% TBE UREA polyacylamide gel and then purify the libraries on a 5 % TBE polyacrylamide gel selecting between 135 and 150 bp to get a clean result.
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Old 09-01-2015, 05:51 AM   #13
chariko
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Quote:
Originally Posted by huguesparri View Post
We used Spin-X from Costar and they worked fine.
Hi Huguesparri,

I checked Spin-X from Costar (http://www.sigmaaldrich.com/labware/...ePage=17192542) and they only have 0.22 or 0.45 um filter tubes. Illumina´s recommendation has 5 um.
In this step I am just filtering out the gel pieces but I wonder they recommend 5 um in order not to clog the porus thing that could happen in case you use a 0.45 um filter tube. Do you think this could happen? How could you avoid this clogging?
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Old 09-01-2015, 11:01 AM   #14
NextGenSeq
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We find the miRvana kit from Lifetech works better than the Qiagen miRNAEasy kit for miRNA isolation.

The Bioo Small RNA V2 kit uses degenerate adapters which help to eliminate the severe ligation bias seen in the Illumina kits

http://www.biooscientific.com/next-g...ry-prep-kit-v2
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Old 09-01-2015, 04:35 PM   #15
nucacidhunter
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Quote:
Originally Posted by chariko View Post

I checked Spin-X from Costar (http://www.sigmaaldrich.com/labware/...ePage=17192542) and they only have 0.22 or 0.45 um filter tubes. Illumina´s recommendation has 5 um.
In this step I am just filtering out the gel pieces but I wonder they recommend 5 um in order not to clog the porus thing that could happen in case you use a 0.45 um filter tube. Do you think this could happen? How could you avoid this clogging?
NEB kit recommends using 0.45 µm gel filtration columns (same as the one you are pointing) with the same gels as recommended by both Illumina and NEB. They instruct to centrifuge the filter for 2 min @13.2k rpm.
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Old 09-02-2015, 09:36 AM   #16
Olaf Blue
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We use these to clean up gel purifications:

http://www.emdmillipore.com/US/en/pr...M_NF-UFC30GVNB
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Old 04-17-2018, 12:34 PM   #17
MarylandKR
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Default Zymo Select A Size

Does anyone have any experience using the Zymo Select a size instead of gel purification?
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