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Old 12-12-2012, 06:36 PM   #21
tanals
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Location: Singapore

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Hi Katherine

Thanks for your responses. I've basically done pretty much as you did and still got the same results. I did run an extraction without the DNase step (either on-column or solution based, which are part of either Qiagen's micro or mini kit) and I'm pretty sure that the salt contamination comes from the wash buffer here. As our facility will not accept samples unless the A260/230 ratio passes their QC, I'm going to try a set without DNase treatment and see how that goes. Your trizol protocol may be worth a go, do you mind posting it too? I'm using this with phase-lock gel tubes, have you ever tried those?

Thanks
Tanals
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Old 12-13-2012, 08:33 AM   #22
Katherine.Waugh
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Hi Tanals,

I actually started with a TRIzol protocol that included the phase lock tubes. It is written below. The DNase treatment was very important for me because a lot of my 260 reading was coming from DNA, and I thought I had a lot more RNA than I really did. If you treat it on the column it avoids losing RNA.

Our facility told me the same thing, but I just said "I optimized my protocol by looking at the nanodrop curves and ratios, then used the Qubit to quantitate because it is more accurate for my tiny amounts of RNA" then submitted that with my bioanalyzer trace's and RINs. They took the samples without question, then, but I just submitted them so I can't tell you if it worked for the microarray (switched to nugen amplification and affymetrix microarray because of low amounts of RNA, time of processing for our grant we want to submit soon, and experience of our facility).

One thing that can effect your ratios with a TRIzol extraction is the phenol so be careful if you think it is salt because you don't know that for sure. Phenol generally gives you a pretty specific curve, though... A good example that helped me is in that paper I referenced early on in this thread. I found the shaking really well after having it frozen is very important for proper separation.

The phase extraction (that, in my hands, couldn't get rid of the DNA anyways) combined with the DNase treatment seemed like just extra steps that I could be losing my RNA at. When I switched to the RLT lysis I got more and higher quality RNA. I'm sure it's different in everyone's hands, but the switch really helped me. Maybe a neighboring lab has some you can try before buying your own? TRIzol is also not the best for very small sample sizes. It was ok until I got down to about 50,000 sorted lymphocytes by flow cytometry, or 20,000 spleenocytes that were counted by microscope and then put into the tube (my practice samples like this likely had more cells than my sorted lymphocytes). Every cell type is different, though, so you will have to see for your cells.

TRIzol protocol our facility gives us and that I optimized in my hands for my small samples:

Note: This wasn't as good as the RLT protocol above if you can change.

Tips similar to before:
-Do not vortex as this could shear long RNA or DNA so that it is harder to detect.
-Make EtOH fresh.
-DNase sensitive.
-Centrifuge mode off of "soft" if have it.
-Get everything ready and RNase-zapped ahead of time.
-Low binding tubes
-Discard flow through from wash steps by pippetting to reduce salt contamination.

Protocol:
1) Sort cells, spin down, aspirate off supe (as before), and resuspend in 1 mL TRIzol.
2) Store -80 for up to 1 month
3) Thaw quickly at 37 degrees Celsius. Important sample is at room temp before phase extraction.
4) Mix really really well by pipetting and shaking vigorously or phenol will stay in aqueous phase. This was something that was really messing with my nanodrop curves and readings so mix really well.
5) QIA shredder to homgenize and pellet down crap/protein as before
6) Meanwhile, spin phaselock gel tubes in a separate centrifuge at 16K xg for 1 minute. Position tubes so all handles facing outwards.
7) Add 250 uL chloroform to samples before adding to phase lock tubes. Shake vigorously ~15 seconds.
8) Add sample with chloroform to phase lock gel tubes. Let sit room temp 3 minutes.
9) spin 16K xg at 4 degrees Celsius for 5 minutes.
10) Add 250 uL chloroform again. Invert gently to mix. Let sit room temperature 3 minutes.
11) Spin 16K xg at 4 degrees Celsius 10 minutes
12) Transfer top clear layer to new tube
13) Let sample and RNeasy columns come back to room temperature for ~1-2 minutes before continuing on.
14) Add 70% EtOH 1:1, shake to mix.
15) Bind samples to column by transferring to Qiagen RNeasy miniElute column (Cat # 74204)
16) Spin 10K xg room temperature for 1 minute. Do sample in two parts to fit into column.
17) Discard flow through. Repeat for remaining samples.
18) DNase treat on column as specified in RLT protocol above.
19) Wash with RPE 2x and 80% EtOH 1x as specified in RLT protocol above.
20) Finish same way as with RLT protocol above.

Good luck again!

Katie
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