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Old 09-27-2012, 12:15 PM   #1
Katherine.Waugh
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Question RNA quality for library generation: Low 260/230 ratio

Hello,

I am having issues isolating quality RNA for gene expression profiling.

I isolate total RNA from samples stored in Trizol followed by an extra purification step via Qiagen RNeasy Minielute columns (Cat. # 74204). I obtain small amounts (20-240ng) of RNA according to a nanodrop with a RIN number greater than 9 on the bioanalyzer.

However, on the nanodrop I get a peak a 230 (charcterisitc "shoulder" at 225nm), and a bump and 270nm. I have heard that these two peak problems are normal when isolating small amounts of RNA via Trizol and near impossible to remove.

I would like to use the RNA to make libraries for gene expression profiling on the Illumina HiSEQ but do not want to submit low quality RNA. Is there a way that I can check ahead of time to make sure that whatever the source of these peaks will not affect library construction (RT-PCR?).

I would greatly appreciate any ideas on how to further purify my RNA (butanol/ether experiences?), any past experiences as to whether or not such nanodrop readings predicted poor library construction, and other ways to better purify small samples already stored in Trizol.

Thank you for your time,

Katie
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Old 09-27-2012, 10:05 PM   #2
upendra_35
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Hi Katie,
How is your RNA look like on bioanalyzer? Looking at your RIN number, it seems to me that your RNA is good for library preparation. Also don't worry about sending the RNA to the sequencing facility as they will look at your bioanalyzer traces and let you know if the RNA looks good for sequencing or not.
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Old 09-28-2012, 09:12 AM   #3
Katherine.Waugh
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Default Nanodrop "quality" required for facility submission

Hi Upenda,

Thank you so much for the response!

My RNA looks normal on the bioanalyzer. However, if I cannot get my RNA "quality" to appear better on the nanodrop through protocol suggestions from the Qiagen technical support, do you know of a couple of less-expensive experiments that I can try on practice samples of RNA isolated in the same manner to see if the possible contaminants will effect library construction? I am planning on doing RT-PCR (to see if generating cDNA or cDNA amplification is effected), but I am concerned that the only way to truly see if the cause of the unusual nanodrop curve is going to hinder library construction is to just invest in trying to actually generate libraries from a couple of the samples themselves. What are your thoughts on this?

Thank you again for the help!!!

Katie
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Old 09-28-2012, 10:52 AM   #4
drosoform
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Dear Katie,

I have also isolated small amounts of RNA (50-300 ng/ul) with the Qiagen RNeasy Mini kit, and would often get low 260/240 ratios (around 1 or even less), no matter how careful I was to avoid getting Trizol and other junk in my samples.

We went through a lot of trouble to improve our protocol. Perhaps you have seen these before, but here are some tips that helped us a lot:

- Make sure Buffer RLT and centrifugation steps are done at room temp (it may crystalize if too cold)
- When washing, roll the column around to make sure the entire inside gets washed

(as seen at: http://answers.yahoo.com/question/in...6155235AAi9J1h)

After doing these things, we saw great improvement in 260/230 ratios (now 1.8-2).

Also, for qRT-PCR, we have recently switched to a non-Qiagen RNA isolation, the older way where RNA is purified with 3M sodium acetate and the pellet is washed with ethanol. This method has been very successful for us and gave us higher total RNA yield as well as consistent good purity, at least from Nanodrop. It is much less expensive compared to the Qiagen method. We haven't done any HTS stuff with this method to isolate RNA yet, but maybe it is something you might consider trying?

Good luck!
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Old 09-28-2012, 02:22 PM   #5
Katherine.Waugh
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Default Cleaning-up small amounts of RNA for Illumina RNAseq

Dear Drosoform,

Thank you so much for the reply. Your comments confirmed the following of what the Qiagen tech support said:

"With regards to your query below, low 260/230 ratios are usually due to carryover of small amounts of guanidine thiocyanate, often in combination with very low RNA concentration. I would also suggest the following:
1) Performing multiple RPE wash steps to remove residual salts.
2) Perform slightly longer and slightly faster centrifugation steps.
3) Ensure that the procedure is performed at room temperature (please do not place samples on ice or spin at 4 deg C).
4) Ensure that the sample is not too cold prior to addition of Buffer RLT.
5) Ensure that the RLT buffer does not contain any precipitates. If there are precipitates, equilibrate the buffers at room temperature prior to use.

They then went on to say in an attached newsletter the following: "In our experience, increased absorbance at 230 nm in RNA samples is almost always due to contamination with guanidine thiocyanate, a salt which absorbs very strongly at 220–230 nm and is present at very high concentrations in the lysis buffer or extraction reagent (e.g., TRIzol®) used in most RNA purification procedures. Our experiments show that the A260/A230 ratio of an RNA sample is strongly reduced when guanidine thiocyanate is present even at submillimolar concentrations (Figure 6A). However, we also found that concentrations of guanidine thiocyanate of up to 100 mM in an RNA sample do not compromise the reliability of real-time RT-PCR, even when using PCR chemistries that are sensitive to inhibitors (Figure 6B). Similar observations have been reported by other researchers (2)."

However, after optimizing my RNeasy purification with suggestions from the tech support and your post I was not able to improve my 260/230 ratios. I think this is because the extra RPE washes dramatically reduced my RNA yield.

I may try your suggestion to purify with 3M sodium acetate and wash with ethanol, and I may also try a butanol/ether purification that I found in the following paper: "A simple and loss-free method to remove TRIzol contaminations from minute RNA samples" Helmut Blum et al 2008.

However, I may just use my protocl as is after speaking with some local PI's they have said they constructed RNAseq libraries from samples with similar nanodrop curves. Upon further inquiry Qiagen tech support has said " If you have a very small amount of RNA, this will skew the 260/230 values, no matter how many times you try to clean it up. You can try to perform ethanol or isopropanol precipitation, but your RNA pellet will be very small and hard to see. I would suggest that you use the RNA for the downstream application already."

I feel that the best thing to do would be to quantitate my samples again via Qubit, see if an RT-PCR works, and if it the sample has enough RNA and works for the RT-PCR I will continue with Illumina library construction to see if whatever is contaminating my quality RNA hinders quality library construction.

Thank you for your help, and I will keep this thread updated on whether or not the RT-PCR and library construction is successful. If it is not successful I will keep this thread updated on which methods, in my hands, have worked to further purify small amounts of RNA stored in TRIzol for such future applications.

Thank you again for your time!

Katie
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Old 09-29-2012, 12:00 PM   #6
pmiguel
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Hi Katie,
The 270 nm peak is from the phenol in Trizol. The 230 nm peak can be various substances, but given that Trizol contains guanidium, it probably is a salt thereof. However, acetate (eg, from sodium acetate) can also absorb in that area. More details about various contaminants that absorb in the UV here.

I always advise people doing RNAseq to do a final DNAse treatment followed by a column clean up. I like the this kit for that. However, like many other columns of this type, you can get a little salt carry-over that gives you some UV absorbance around 230 nm. It probably means you have not quite gotten the protocol down perfectly. But as long as the peak isn't sky high, the salt concentration is probably not going to interfere with the first step of the TruSeq RNA prep kit.

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Old 09-29-2012, 03:22 PM   #7
Katherine.Waugh
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Hi Phillip,

Thank you for the response. I have been very concerned about the 270nm peak and even switched to Phase Lock Gel tubes for the phenol chloroform extraction just in case my technique is to blame. However I still cannot get rid of the little extra bump at 270nm. The only way that I have been able to get rid of the abnormal nanodrop RNA curve is to increase my RNA concentration (cell number) with the same protocol. It is my belief that the contaminant at 270nm is still there but masked by the larger 260 curve in this case. Do you have any other suggestions as to how I might optimize my protocol to get rid of the possible phenol? I am very open to suggestions and would like to try everything possible before touching my precious samples!

My 230nm peak isn't sky-high, but relative to my 260nm peak it still significantly lowers my 260/230 ratio. The curve actually looks very similar to Figure 1A in the paper that I cited earlier (A simple and loss-free method to remove TRIzol contaminations from minute RNA samples Helmut Blum et al 2008).

I have incorporated a DNase treatment on the RNeasy columns. I have heard that without this people have had up to 40% of their reads map to introns with Trizol-based isolation procedures and am trying to be as careful as possible!

Thank you again,

Katie
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Old 09-29-2012, 03:25 PM   #8
Katherine.Waugh
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Default Dear Drosoform-

I mis-typed earlier: I isolate a maximum of 20-40ng/uL of RNA. Have you worked with lower than 50ng/uL RNA? I am so sorry for the earlier typo.
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Old 09-30-2012, 07:24 AM   #9
pmiguel
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Hi Katie,
Might help your sense of well-being to calculate what concentration of phenol your preps contain. I think you will find that it is very low and unlikely to affect downstream assays -- except for UV spectrophotometry.
But I am surprised you would have phenol carry-over after running a RNeasy column, even at the low level to make it detectable via UV spec.
In any case I think you want to think about your QC assays in a different way. Your samples could be contaminated with all manner of substances -- as long as they don't absorb in the UV, you will be blissfully unaware. So it doesn't make sense to fixate on any one QC assay too much. When I see RNA samples that have a little phenol left over, I focus on the nano chip result. If that looks good for total amount of RNA, I declare victory and move on.
The concentration of RNA you mention does sound low, but as long as you are over 1 ug of total RNA, you will likely be fine. The thing that will screw up the first step of TruSeq RNA is stuff that would interfere with hybridization of the polyA RNA to the oligo dT, etc.
By the way, I did not look at the phenol clean-up method you invoke, but if it is the butanol/ether one -- sure, that should work fine. Just remember to water saturate your butanol prior to extractions unless you want to reduce your volume because it will pull some water out of your samples. (We used to use non-water-saturated butanol as a method to concentrate aqueous solutions.)

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Old 10-01-2012, 07:51 AM   #10
Katherine.Waugh
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Hi Phillip,

I really appreciate your replies and will keep this thread posted as to how my library prep goes!

Thank you,

Katie
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Old 10-16-2012, 07:44 PM   #11
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Hi Kathie and everyone,

Any new insights into the low 260/230 ration problem using Trizol? I have identical problem: small sample size> small amount of RNA (which is OK) and very low 260/230 ratio (less than 0.1).

Thanks a lot,
Guy
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Old 10-17-2012, 07:48 AM   #12
Katherine.Waugh
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Hi Wacguy,

I started quantitating my samples with a Qubit as the nanodrop that I was using should only be accurate down to ~20ng. I also did RT-PCR to see if the contaminant(s) at 230nm would inhibit the reaction (it did not). I have not yet tried to construct libraries for RNAseq to see if the contaminant inhibits this but can keep you posted! I still also check quality with a Bioanalyzer and routinely get samples with RIN > 9.

I am planning to use these RNA samples that I have isolated with TRIzol, but for samples that I am isolating now I have switched to Qiagen's RLT plus + BME. The RNA appears much more clean on the Bioanalyzer with this switch. Also, my samples always look better when processed fresh than stored in lysis buffer if you can process them right away. Are you storing your samples in TRIzol? If so, are you mixing really really well upon thaw (otherwise phenol can isolate in the aqueous phase) and letting the TRIzol come to room temperature before phase separation?

If you would like, I can post my TRIzol and RLT plus protocol here that I have had the most success with the little tricks that have reduced salt contamination in the RNA sample. How much RNA are you working with? From what source? What is your isolation protocol?

Katie
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Old 10-17-2012, 08:35 AM   #13
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Hi Kathie,

Thanks a lot for such fast reply. I'm new to RNA world. I used Trizol and no kit to get all RNA types. I ground my small piece of root w/ liquid nitrogen and added the Trizol; (do you think I should wait a few secs till the ependorf is at RT w/ the powder and then add the Trizol?). I don't store the samples in Trizol. After adding it, I add 1/5 of a volume of Chloroform, centrifuge and transfer the aqueous phase, add Glycoblue and precipitate w/ ethanol and NaAc O/N. Next day I centrifuge in 4oC for 30' and wash twice w/ 75% EtOH and then dissolve in water. I got 5-10 ng/micrl (w/the nanodrop) which was surprisingly high but as I mentioned, extremely low 260/230 ration. Qbit was less friendly to such low RNA concentrations. Did you try the Butanol method; I'm considering of trying it but we don't have ether here although Butanol can be discarded w/o the ether I think: (http://www.ncbi.nlm.nih.gov/pmc/arti...00185-0196.pdf). I'd love to read about your tricks, maybe I can skip the Butanol part that seems a bit tedious to me.

Thanks a lot again,
Guy
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Old 10-17-2012, 09:17 AM   #14
Katherine.Waugh
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Hi Guy,

The Qubit is more specific for RNA than your 260nm reading on the Nanodrop. Have you considered incorporating a DNase treatment? In my experience, it is really hard to not get DNA contamination with TRIzol extraction and this can contribute to your 260nm reading and make you think you have more RNA than you do.

I did not try the Butanol method for the same reasons you mentioned. However, if you don't need to get rid of whatever the contaminant is being detected at 230nm (salt from TRIzol?) I wouldn't mess with it. Have you tried your downstream application yet? Have you looked at your sample quality via Bioanalyzer yet? How does that look? Have you tried dedicating more of your sample to the Qubit reading? The one that I have access to can detect as low as 5 ng of RNA so I usually use 2 uL of my samples to quantitate. I discovered I had much less than 20 ng/uL of RNA after DNase treatment. You can test for DNA contamination in your samples that have not been treated with DNase with an RT-PCR +/- the reverse transcriptase.

I don't think you need to wait with the powdered sample before adding the TRIzol. Just make sure that the TRIzol+sample is at room temperature before the phase extraction.

My tricks are basically modified versions of a Qiagen column clean-up kit with an on-column DNase treatment. However, they never gave me very high 260/230 ratios. I think this is because my sample was too small for an accurate nanodrop reading. As my 260 value decreases and my 230 value stays the same it's really effects the ratio. I have decided to just move forward with my downstream applications and see if it will work as my RNA is high quality. I will post my TRIzol and RLT protocols later tonight.

Good luck in the meantime!

Katie
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Old 12-10-2012, 04:17 AM   #15
tanals
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Hi Katie,

Just wondering if you ever managed to get your RNAs to work? I have the exact same problems with mine, A260/230 <0.5 even though my RNA concentration and absolute yield are workable (although still <10ng/uL). Would be massively grateful if you would post the modified RLT protocol? I'm also using the RNeasy Plus Micro kit, with multiple cleanups and concentration steps, but with no significant improvement to the ratio (also no improvement to my A260/280). Quite frustrating!
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Old 12-10-2012, 05:31 AM   #16
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Default RNAzol

Hi Tanals,

I'm using ENAzolRT and it gives really good results; high concentration of RNA (in respect to the small tissue size I am starting with) and good 260/280=1.7 260/230=0.8 values, not perfect. The RNA was tried for qPCR w/ 3 primer sets and there were no problems so I guess it is OK.

Guy
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Old 12-10-2012, 05:35 AM   #17
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For the record, we rarely look at Nanodrop readings for our library preps.
Quantification information from it is often thrown off by any increased A270 values. We either use the Qubit or the Bioanalyser. The Bioanalyser isn't brilliantly accurate, but as long as we can get >100ng based on it's reading we seem to produce more than enough library. RIN's on the Bioanalyser are important, however, as you need non-degraded transcripts to avoid 3' bias.
You also have to remember, the first steps of the TruSeq RNA protocol is a mRNA isolation on Oligo dT beads. This in non-enzymatic and includes an ethanol wash, which should remove any left over nasty stuff in your extractions.
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Old 12-10-2012, 06:20 AM   #18
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Man, I wish no one had ever invented the idea of checking two 'diagnostic ratios' with UV spec. Seems to just encourage people to ignore the full spectrum that the nanodrop gives them and concentrate only on these (often meaningless) metrics.

UV spec is a powerful tool but it seems to have accumulated too much protocol 'baggage' from the 1960's (or whenever) to be of much use these days. C'mon guys, think for yourselves. If you want to use a nanodrop, take a few of the UV-absorbing substances in the lab (acetate salts, phenol (@ 0.1% in water), guanidine salts, beta mercaptoethanol) and run spectra on them. Hang them by your desks so you can learn them as well as your mother's face.

While you are at it, calculate 260/280 and 260/230 values for each so you can see how easily these common lab compounds can confound this assay. Here, let me lay down the guantlet -- until you do this you are not worthy to use a UV spectrophotometer. At best you are an automaton malfunctioning every time you are given a sample that is just a little outside of perfection.

Or, you can read my post where I ran some of these spectra. Perhaps that will suffice. Better yet, find some other UV absorbing compound and post its spectrum to that thread!

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Old 12-10-2012, 08:57 AM   #19
Katherine.Waugh
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I found it most useful to look at the nanodrop curves, bioanalyzer trace and RIN, qubit concentrations, and RT-PCR +/-reverse transcriptase while I optimized my protocol. I also started to work with a technician that processes hundreds of RNA samples a week that have been stored a variety of ways. She gave me tips from samples that are always her favorite to process. My protocol and tips are written below.

I am submitting my samples for microarray + nugen amplification because I could only get a 5 ng total RNA for my smallest samples. I will let you know if it worked once I find out!

RNA extraction: RLT plus (Qiagen #11048449) +BME version

Tips:
-Use of RLT plus gives me more RNA and much more clean RNA instead of TRIzol
-Processing fresh is best for the above reasons as well. If freeze, do a quick thaw at 37 degrees Celsius
-Make EtOH fresh. I make sure that it's less than one week old. Some prefer to make it that day.
-DNase is very sensitive to physical denaturation so be very careful (gently invert only to mix, no vortex or pipetting up and down)
-If centrifuge has the option, put centrifuge "soft" mode OFF as many of the spins are short. This helps get more RNA-looking nanodrop curves (by getting rid of contaminants more)
-Get out all tubes and reagents (RNase-zap these, pipetts, and hands) before begin. Don't get too crazy as can hurt RNA too! Make sure to let all dry or kim wipe off at least before working with it. Getting out the RNeasy tubes now is important too as all of the steps with this are supposed to be at room temp and not being so can effect isolation.
-Low binding tubes and tips are good so don't lose any of precious RNA isolated.
-Discard flow through by pipetting to reduce salt contamination on outside of RNeasy column that could contaminate at later time.

Protocol:
1) Sort cells, pellet down 350 xg 20 mins, aspirate off supe, resuspend in 1mL RLT plus (Qiagen #11048449) + 10 uL BME (freshly mixed by vortexing together before adding to sample). Can look-up and optimize how much RLT+BME to add, but is a lysis buffer. I went for the over kill with my 30K sorted cells to keep it standard across my samples. Only danger I could think of with this step is not using enough lysis buffer. RLT plus got better reviews by the technician cited above than the straight RLT that comes in the RNeasy kit specified below.

2) Mix really well by vortexing sample on max speed for 30 seconds - 1 minute. Don't bother pipetting up and down here or at later steps as just gets RNA into tip and potentially lost. Some will say not to vortex here as can shear long RNA and DNA so harder to see if have contaminating DNA by PCR. I optimized not having contaminating DNA without the vortex step (shaking vigorously instead), then switched to this to get maximum lysis (and saw max amount of RNA isolated with vortexing).

3) Spin down 10K 1 minute to get all of sample back down to bottom of tube. Can store in -80 here if need to, but I like to process fresh and saw best results if just did it all now.

4) Pipett to QIAshredder (Qiagen # 79654) to homogenize and pellet down crap (proteins, etc) to be avoided when transfer to new tube. Spin down 2.5 minutes on max speed at room temperature. I like to circle where all of the protein will pellet down to so I know to avoid that part of the tube when aspirating up everything I can with my pipett on the other side. I use the same tube with the same spot facing towards the outside so I know where to avoid. I used to not do this step, but it did help make everything a little bit more clean.

4) Transfer to new tube. Again, I like to use siliconized tubes (Low bind) to avoid RNA loss.

5) Add 70% EtOH 1:1 (1 mL here) and shake vigorously about 15 seconds to mix. I kept the shake instead of vortex here because I didn't see a difference with vortexing at this step and tried to avoid vortexing for reasons specified earlier. If use 1 mL, will likely do your sample in two tubes. half in one, half in another. A common mistake it to not take the exact proof of the EtOH stock into account when making your 70% EtOH. The one I use is 200 proof molecular biology grade. Make sure you're really working with 70% EtOH and not lower.

6) Quick spin to get your sample back down to bottom again.

7) Bind samples to column by transferring to Qiagen RNeasy miniElute column (Qiagen # 79254). Spin 10K xg at room temp for 1 minute. I like this column because of the very small elution volume (14 uL) and the subsequent concentration of my RNA.

8) Discard flow through by pipetting, not decanting. This just got my RNA curves a little bit better by reducing salt contamination on the outside of my column that spins down at later steps.

9) Repeat steps 7-8 until all of sample has been run through column.

10) DNase treat on column (Qiagen RNase free DNase set recommended by RNeasy column packet #79254) according to directions and repeated below:
-Wash column with 350 uL RW1 from RNY ME kit at 10K xg 15 seconds.This doesn't come in any of the Qiagen catalogue numbers specified above and I found it in another kit we had lying around. I put RW1 directly on column instead of running down sides to avoid having salt from RW1 at higher column areas. This is very salty, I think, based off of nanodrop curves (see below reasons in RPE wash step).
-Add (10 uL DNase + 70 uL RDD mixed by gentle inversion if any) to the column. Do not pipet up and down as can hurt DNase.
-Incubate at room temp 15 minutes
-Wash with 350 uL RWI again and spin 10K xg 15s
-Can repeat DNase treatments or increase incubation time to 20 mins if necessary. I optimized this in my hands by doing RT-PCR +/-reverse transcriptase to get rid of contaminating DNA. One treatment at 15 mins was sufficient and it's what I do for now on. My PCR method could only detect down to 0.1 ng DNA, though, as I saw by running specific amounts of DNA in other gel wells to know how low I could detect with this method.
As people said above, though, you can get rid of DNA in the mRNA selection for the RNAseq... but without DNase treatment you might think you're looking at a lot more RNA than you are. I kept it for accurate quantitation so I knew exactly which gene expression profiling platforms were options for me.

11) Wash column with 500 uL RPE at 10K xg at room temperature for 1 minute. Make sure to invert (and roll between fingers) RPE on column for ~2 minutes before spin to get rid of all possible salt still stuck to tube. For inversions, put into fresh tubes to prevent getting old liquid/salts on the outside of the RNeasy column that could contaminate at later time/spin. Some like to increase the RPE amount to 700 uL. If have a lot of salt contaminants (230 nm UV, can do this step multiple times. With the RW1 wash needed for the DNase treatment, I found it was very salty and I needed to do two RPE washes to get rid of it. I only needed to do one RPE wash without the DNase treatment.

12) Repeat RPE wash step 11. See above. I still put it into a fresh tube for the inversion here again.

13) Wash column with 500 uL 80 % EtOH, spin 10K xg at room temperature for 1 minute. Some have optimized their isolation to get rid of this step. I haven't seen a difference with or without it either, but have kept it.

14) Transfer column to new tube.

15) Spin with top open to dry for 5 minutes at 16K xg at room temperature. Any hint of EtOH and won't pull off RNA.

16) Transfer column to new tube. Elute RNA by adding 14 uL RNase free water to column. Incubate at room temperature 1 minute with lid closed and spin 16K xg for 1 minute.

17) Repeat step 16 by using the 14 uL flow-through. I got twice as much RNA this way sometimes.

18) Inspect RNA samples via nanodrop (UV ID of contaminatns and quantity estimate if have enough RNA for this. I liked it to see if my curves looked like normal RNA curves.), bioanalyzer (quality of RNA/degredation), qubit (quantity if need specific and can do as low as 5 ng/multiple uL), and RT-PCR (+/-DNA contaminants). I did all of these while optimizing my isolation with practice samples of the same number of cells that I had for my samples, but now with my real samples I just do bioanalyzer. If that estimates that I can quantitate by qubit and not waste all of my sample in the process I do that. If it doesn't, I just use the bioanalyzer estimate and will see how the microarray core wants to proceed from there.

Please let me know if any of this isn't clear. I have a similar TRIzol isolation protocol too if you would like me to post this. This is basically just a slightly modified version of the Qiagen RNeasy column and DNase treatment protocols... but with specifics added in that tech support, other labs, and personal experience has let me add in. Good luck!!!

-Katie
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Old 12-10-2012, 08:59 AM   #20
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PS. Using RLT plus vs. TRIzol and isolating fresh are two things that have started to give me RIN values of 10 every single time. My only ones lower than that were not done fresh and/or processed with TRIzol. Good luck again!

-Katie
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