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Thread | Thread Starter | Forum | Replies | Last Post |
cDNA prep for Illumina using the Nextera kit | fgoetz | Sample Prep / Library Generation | 3 | 07-09-2012 06:34 PM |
Alternative to Illumina Paired-End Sample Prep Kit | Sarah_Tulin | Sample Prep / Library Generation | 0 | 12-12-2011 12:00 PM |
Problems with Invitrogen Size Select E-gels for Illumina RNA-Seq Library Sample Prep? | Jerry Glenn | Sample Prep / Library Generation | 0 | 04-18-2011 07:14 AM |
Paired end Illumina prep problems? | James | Sample Prep / Library Generation | 4 | 09-27-2010 11:10 AM |
Homemade illumina oligos and library prep kit | elly | Illumina/Solexa | 3 | 03-24-2009 08:05 AM |
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#1 |
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Location: Medford, MA Join Date: Aug 2011
Posts: 11
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Hello all,
I have recently gone through the Illumina Trueseq RNA sample prep kit with no success. Has anyone else had problems with this kit/what were any problems encountered? A little bit more about what I did: I extracted with Ambion's RNAqueous 4-PCR kit but do not have a bioanalyzer to assess total RNA quality prior to the protocol which I have heard can have a huge effect on yields. Instead I ran a 2% agarose gel in TAE and stained with Ethidium. I got one bright, non-smeared band (I am working on Lepidopteran species which have a "hidden-break" in the 28S band, so it migrates with the 18S band) for all of my samples, so I assumed the RNA was ok. I ran 10 uL of sample on this gel with RNA concentration being from .332-1.66 ug/mL. Questions: 1) Would it have been better to run a denaturing RNA gel? I talked to Illumina tech support and they recommended this. Could I see denaturation better? 2) I standardized the total RNA put into the Trueseq protocol to 1 ug... should I have used more starting material? This is on the lower end of the recommended range of .01-4ug. 3) Our department just acquired a nanodrop so I speced the RNA that had been stored at -20C for 2 weeks since I tried the Truseq protocol. The 260/280 readings were from 1.25-2.03. I don't have a lot of experience with these machines but think the range I am looking for is 1.8-2.1 , does this mean that some of my samples are contaminated with excess salts/proteins/something else? Any advice would be appreciated. I know crap in is crap out, so does it seem like my starting material is ok/are there any additional methods I can use to check it? Thank you! |
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#2 |
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Location: Phoenix, AZ Join Date: Mar 2010
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Finding a way to get a bioanalyzer run would be a good idea but in the absence try the denaturing gel only after nanodrop readings meeting the following requirements:
Concentration: 200-400 ng/ul (dilute to range) 260/280: >1.8 min better if >1.9 260/230: >1.9 min better if >2.0 (this one is important, tells if you have contaminating ethanol that will mess up the down stream steps) |
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#3 | |
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Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008
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Ethanol doesn't absorb at 230nm itself, but acetate does. So if you are using sodium acetate as your precipitation salt, then it might show up at 230 nm. But that does not mean that the sample still has ethanol in it (although it might). It just means that the acetate salt precipitated and was not sufficiently resolubilized by the 70% EtOH washes. Also, the first step of the TruSeq RNA kit is polyA RNA purifications. This is hybridization based, so I am not sure a little ethanol and/or sodium acetate would ruin this step. -- Phillip |
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#4 | ||||||
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Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008
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Was your result no band at all after the final 15 cycles of PCR? -- Phillip |
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#5 | ||||
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Location: Medford, MA Join Date: Aug 2011
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Additionally, something that caught my eye is that when we looked at our sample concentrations on a Qubit via RNA assay we got a concentration reading of 1.66 ng/uL and when we used the nanodrop on the same sample we got a concentration reading of 44.6 ng/uL. The nanodrop reading was two weeks after the Qubit but I can't imagine RNA/DNA being created in the -20C in this timeperiod. Would this also indicate some sort of DNA/protein contamination? Best, Crista Last edited by cburke11; 08-02-2011 at 09:24 AM. |
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#6 | |
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Location: DC Join Date: May 2011
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Cheers, T |
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#7 | ||
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Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008
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Fluorimetric assays (eg, ribogreen) will generally not detect mono or oligonucleotides. So you may be starting with way less RNA than you think you are. -- Phillip |
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#8 |
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Location: Medford, MA Join Date: Aug 2011
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I had really low 260/230 readings, is this guanidine contamination and has this been know to affect the Truseq protocol?
The Ambion RNAqueous 4-PCR kit uses a guanidium-based lysis solution... |
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#9 |
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Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008
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Guanidium salts are strong protein denaturatants, or so I have been led to believe. However, given that the first step of the TruSeq RNA prep involves a hybridization of oligo dT/ magnetic bead purification, it seems likely anything other than very high concentrations of inhibitory salts would be washed away.
I have seen cases where 230 nm absorbing ions (eg, high concentrations of acetate) were concentrated enough that the 260 nm reading was basically a shoulder off the 230 nm peak. That would explain why your fluorimetric reading was so much lower than your spectrophotometric reading. If you presume the 1.66 ng/ul fluorimetric reading was correct, how much total RNA did you use? -- Phillip |
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#10 |
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Location: Medford, MA Join Date: Aug 2011
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I have been using 1 ug which is in the range that is specified to work in the Truseq protocol which is from .1-4ug total RNA.
How does the RNA bind to the beads? If there is a really strong denaturant would there be a protein adapter that is inactivated? |
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#11 | ||
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Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008
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Still, if you live near a zoo, or in certain parts of Africa, zebras hooves may be just as common as horse hooves. And, there is nothing to say you didn't get run over by both horses and zebras. -- Phillip |
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#12 |
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Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008
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#13 | |
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Location: Medford, MA Join Date: Aug 2011
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#14 | |
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Location: Medford, MA Join Date: Aug 2011
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I sure hope I don't get run over by both, one horse or zebra seems to be enough to be trampled by!! |
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#15 |
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Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008
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I forgot to ask:
How long did you do the RNA fragmentation step for? What method did you use to heat the samples? -- Phillip |
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#16 |
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Location: Medford, MA Join Date: Aug 2011
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If you are talking about in the Truseq protocol, the fragmentation takes place with buffers
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#17 | |
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Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008
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From Illumina's TruSeq™ RNA Sample Preparation Guide, Revision A p43, step 1 of "Incubate RFP":
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Typically chemical fragmentation of this sort takes advantage of the catalytic action of divalent cations (eg, Zn++ or Mg++) to accelerate nucleophilic attack of 2' hydroxyl groups on the phosphate backbone, forming 2'-3' cyclic phosphate, and breaking the strand at that nucleotide. However, without heat (94 oC), very little fragmentation will occur. I brought this up because you may have been like us and wanted longer insert amplicons than the default "150 nt" ones. However Table 12 on page 113 is either misleading or just plain wrong. In it, a 0 minute incubation at 94 oC is said to result in 200 nt inserts. We tried a 1 second incubation at 94 oC to obtain those 200 nt inserts. However upon running the double stranded cDNA resulting from that RNA with very limited fragmentation time we were shocked to see this size distribution on an Agilent High Sensitivity Chip: ![]() Average size was 1200 bp! That is great for people who prefer to sonicate cDNA for their fragmentation step. But you can see that a very small proportion of the cDNA will be in the ~200 bp size range. So I don't know what Table 12 means. I tend to think it is just wrong. Anyway, I ask again: how long did you incubate at 94 oC and what method did you use to heat the samples? -- Phillip |
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#18 |
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Location: Oregon Join Date: Jan 2011
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Hi Phillip,
Did you get to the bottom of the fragmentation time and median insert length issue? I spoke to a lab that uses 6 seconds, but the only person in the lab did not know what size fragment resulted. Thanks for your help! |
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#19 |
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Location: USA Join Date: Apr 2009
Posts: 482
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Use the NuGEN Ovation RNA-Seq kit. Illumina's kits are among the worst out there.
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#20 |
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Location: Oregon Join Date: Jan 2011
Posts: 24
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Thanks for the advice, but I have the TruSeq RNA kit and all necessary consumables in hand. I need to learn how to make this kit work well for us.
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Tags |
rna integrity, rnaseq, sequencing, truseq |
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