miRNA Truseq 5 μm filter tube size alternative

[chariko]

Hi all,
I am planning to run a small RNA Seq (TruSeq Small RNA Library Prep) in a Illumina MiSeq and reading the protocol they use a 5 um filter tube to transfer the elute and the gel debris following a centrifugation. Illumina recommends IST Engineering 5388-50.

I was wondering whether anyone has used another filter size and how it worked. I read that user huguesparri used 0.45 um Corning® Costar® Spin-X® centrifuge tube filters and it worked fine (http://seqanswers.com/forums/showthread.php?t=33987) but I would like to have more opinions. Any other alternatives would also be appreciated.


Thanks in advance

[kerplunk412]

You are just filtering out gel pieces at this point, so really any filter spin column should work. We use and recommend the Corning Spin-X columns and they work well. I have even tried a DNA purification column and it seemed to work fine too. In fact, if your yield is pretty high you can probably even just centrifuge the sample at max speed for 5 or 10 minutes and take of the sup without disturbing the gel (you may need to do this twice).

[nucacidhunter]

I have two comments:

1-Be aware that some of Illumina recommended filter tubes have production defect (invisible hole at the bottom of tube) and you may lose your library. Try the tubes before using them.

2- You may adjust gx and spinning time if you are using filter with smaller pore size otherwise some of liquid may stay behind filter reducing yield.

[chariko]

Thanks kerplunk412 and nucacidhunter for your answers.

I didn´t know about production defect for those Illumina´s recommended tubes, this reaffirms my intention of changing the tubes. Also, thanks kerplunk412, I will give a try to the Corning Spin-X columns and I will tell how it worked.

Thanks!

[ppaus]

Thank for all the info. I'll start a miRNA Illumina lib prep soon and wanted to avoid purchasing the recommended filters as I'll only be doing a few samples. My intention was to use columns we already have in the lab and are not eing used.

Does anyone have any experience using DNA/RNA/miRNA extraction columns and DNA/gel purification columns? We have a few different kits in the lab and am trying to decide which one might be more suitable.

Chariko, did you try other columns and how did it work?

Thank you all

[chariko]

Thank for all the info. I'll start a miRNA Illumina lib prep soon and wanted to avoid purchasing the recommended filters as I'll only be doing a few samples. My intention was to use columns we already have in the lab and are not eing used.

Does anyone have any experience using DNA/RNA/miRNA extraction columns and DNA/gel purification columns? We have a few different kits in the lab and am trying to decide which one might be more suitable.

Chariko, did you try other columns and how did it work?

Thank you all

As I told before I finally tried out the Corning Spin-X columns and they worked perfect for me. Anyway, just because this colums are just for broking the gel and get the DNA through the tubes I think the ones you have at your lab will also work fine as DNA is much smaller than any of your filter tubes.

Good luck

[kerplunk412]

Thank for all the info. I'll start a miRNA Illumina lib prep soon and wanted to avoid purchasing the recommended filters as I'll only be doing a few samples. My intention was to use columns we already have in the lab and are not eing used.

Does anyone have any experience using DNA/RNA/miRNA extraction columns and DNA/gel purification columns? We have a few different kits in the lab and am trying to decide which one might be more suitable.

Chariko, did you try other columns and how did it work?

Thank you all

I have done it a few times with DNA purification spin columns and it worked fine. This step is just to filter out the gel pieces, so anything that allows the liquid to flow through but not the gel pieces should be suitable.

[robertami]

Hi, just a quick message to report that Yes the IST Eng. Filter columns recommended by Illumina are faulty!! A hole at the bottom of the collection tube. I reported it and they gave me a replacement bag. I found faulty tubes in that too! Looking for alternative. It's really annoying and not acceptable

[kerplunk412]

Hi, just a quick message to report that Yes the IST Eng. Filter columns recommended by Illumina are faulty!! A hole at the bottom of the collection tube. I reported it and they gave me a replacement bag. I found faulty tubes in that too! Looking for alternative. It's really annoying and not acceptable

The Corning Spin-X tubes work well but are not cheap. Of course you could always avoid the tedious gel step completely with the NEXTflex Small RNA Seq Kit v3, which offers reduced bias and gel-free or low input protocols. As a disclaimer, I work for Bioo Scientific.

Even if you decide not to go with the Bioo kit, I highly recommend you look for alternative small RNA kits. I have said it before and I will say it again- Illumina's kit is basically the worst out there in terms of bias and cost.