PCR amplification and the number of cycles

I had earlier decided that amplifying 8ul for 9 cycles would be good for my libraries. It turned out that 9 cycles was much to few and I increased the cycle number to 12 as per J~'s suggestion. I have now amplified all 40 samples at 12 cycles (still 8ul) and have just finished bioanalyzing the results. It seems that 12 has worked great for many of my samples, but has under-amplified a few (see below).

Of these samples, the first 4 are under-amplified, the middle 4 are fine, and the last 4 are perfect. None are over-amplified which is great. 

Next I need to pool all of these samples into two lanes as per my earlier plans. The pooling will be done so that there are equal molar ratios of each sample in the final mix. The issue I am having is that J~ and I had talked earlier and said that I should amplify each sample the same number of cycles (12) but at this point, 12 cycles has under-amplified some of them and not others. One option is to amplify the low samples a couple times more to bring them up to par with the rest. I am unsure of whether treating some samples differently will cause any catastrophic failures of my experiment. It seems like it will not as the expression analysis will be standardized by the relative expression of a number of housekeeping genes. I would guess that this should remove any effects of cycle number, but I'm not sure. I'll talk to J~ and report back.

Below is a plot of all the samples spread along the X-axis and the final concentration (ng/ul) that the bioanalyzer reported on the Y. The different colors represent different batches of library prep (batch size = 8) and the different shapes represent the different RNA extraction batches (batch size = 4). The horizontal line is at 5ng/ul.

It looks like there are no batch effects for the most part. The one main exception is in the red batch where the squares (males) are lower than the circles (females). Earlier, I had started with unequal amounts of RNA for that batch and I ended up re-doing the males. It seems that something went wrong with the 2nd prep of those, and I need to re-prep them again. As long as I am at it I will also re-prep some of the others that are below 5ng/ul. 

Cycle number in the TruSeq protocol

The qPCR results showed that 13-14 cycles would be excellent for my libraries (see this post). However, the amount of library that I started with was 1 40th of the total library (i.e. 0.5uL). If 0.5ul achieves the exponential phase in 13 cycles, then 8uL should achieve exponential phase in 9 cycles:
starting      cycles to
   ul         exponential
0.5              13
1                 12
2                 11
4                 10
8                  9
16                8

Thus I used 8uL of my libraries and amplified them for 9 cycles. This resulted in some libraries that were perfectly amplified and some that were not amplified enough. The top sample is great, a small even peak (and no secondary hump under the 1500 marker). The second sample does have a peak in the right area, but hasn't been amplified enough - there is not enough library there to sequence.

Fortunately, I only used 8uL of my pre-amplification library, not the entire 20uL just in case this issue arose. So now, instead of going back and re-preparing the entire library, I can just take and amplify the remaining parts. 

I'll need to talk to J~, as there are some serious batch effects (the first 8 all look like the top image, while the second 8 all look like the bottom image) but it looks like 8ul at 11 or 12 cycles will work fine for most everything without over-amplifying the library. As I will be comparing transcript levels, it is important that all the libraries are amplified the same number of cycles. 

Over-amplification is also bad because at that point in the PCR, the DNA molecules are being heated and cooled without being replicated. Thus they will fall apart and re-anneal repeatedly. They will often re-anneal with a DNA of a different sequence. This happens because the adapters have the same sequence, while the center sequences vary, two different molecules can meet eachother and the adapters on the ends will anneal while the middle region will for a big loop. This will mess up the sequencing machine and you'll get bad results. You know if you've over-amplified a library because there will be a low hump that around the 1500 marker. This is the weird hybridized molecules which travel slowly through a gel and so look quite large. In fact they are not any larger than the rest of the library and so can't be selectively removed via their size. The only thing that remains at that point is to re-amplify the library.

In short, I need my samples amplified more, but not too much more, so a cycle or two should be fine. J~ will be able to help me out with that. 

qPCR and strand-specific TruSeq protocol

The qPCR results of my ssTruSeq prep (Sultan, 2012) showed up a little weird:

The group of 4 that amplified first was the females (prepared on 12-15 with []'s of 512, 367, 553, and 455ng/ul from the bioanalyzer) while the second group of 4, amplifying around 14 cycles is the males (prepared on 12-7 with []'s of 3086, 5631, 1783, 2363ng/ul from the bioanalyzer).  The initial concentrations shouldn't matter much because I dilute them down to have 2ng of RNA entering the ssTruseq protocol. In this case though, it seems that I over-diluted the males causing them to amplify later. There is something really odd going on here because when I looked back through my lab book, the concentration of those samples from the nanodrop was much less: 1351, 1559, 1027, and 1276ng/ul. In fact these concentrations are ~4x less (i.e.: two pcr cycles different...). As they amplify on average 2 cycles later, I took a look back at the bioanalyzer results and found that the concentrations were slightly outside the optimal range and therefore likely a little spurious.

In the future I will dilute my samples down and then nano-drop them before I start the ssTruSeq protocol in order to avoid starting with different concentrations of RNA.

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Sultan, M., S. Dökel, V. Amstislavskiy, D. Wuttig, H. Sültmann, H. Lehrach and M.-L. Yaspo. 2012. A simple strand-specific RNA-Seq library preparation protocol combining the Illumina TruSeq RNA and the dUTP methods. Biochemical and Biophysical Research Communications 422:643–646. Elsevier Inc.

RNAseq Samples

Here is the running list of my samples for RNAseq, their RIN numbers, concentration, and library prep. I will keep updating this as I go until I have all 40 samples extracted and the libraries prepared.

#	Date extracted	ID	RIN	[ ] ng/uL	Library
1	9-28-12	BB.BB15.3M	9.6	1071	Yes
2	9-28-12	BB.SS70.3M	9.6	1127	Yes
3	9-28-12	SS.BB20.2M	9.7	859	Yes
4	9-28-12	SS.SS82.1M	9.8	1543	Yes
5	9-28-12	BB.BB15.4F	9.4	1690	Yes
6	9-28-12	BB.SS70.2F	9.4	1427	Yes
7	9-28-12	SS.BB20.6F	9.6	1818	Yes
8	9-28-12	SS.SS82.2F	9.6	1808	Yes
9	9-29-12	BB.BB77.2M	9.3	3086	Yes
10	9-29-12	BB.SS70.5M	9.4	5631	Yes
11	9-29-12	SS.BB25.5M	9.7	1783	Yes
12	9-29-12	SS.SS87.1M	9.5	2363	Yes
13	12-15-12	BB.BB77.1F	9.4	512	Yes
14	12-15-12	BB.SS70.4F	9.3	367	Yes
15	12-15-12	SS.BB25.3F	9.4	553	Yes
16	12-15-12	SS.SS87.3F	9.6	455	Yes
17	12-15-12	BB.BB86.2M	9.4	449	Yes
18	12-15-12	BB.SS72.2M	9.6	483	Yes
19	12-15-12	SS.BB25.3M	9.3	326	Yes
20	12-15-12	SS.SS88.1M	9.6	440	Yes
21	12-15-12	BB.BB86.1F	9.1	235	Yes
22	12-15-12	BB.SS72.1F	8.9	354	Yes
23	12-15-12	SS.BB29.1F	9.1	266	Yes
24	12-15-12	SS.SS88.3F	9.5	507	Yes
25	12-17-12	BB.BB87.6M	9.1	466	Yes
26	12-17-12	BB.SS73.3M	8.6*	466	Yes
27	12-17-12	SS.BB20.8M	9.0	354	Yes
28	12-17-12	SS.SS89.2M	9.0	254	Yes
29	12-17-12	BB.BB87.1F	9.0	411	Yes
30	12-17-12	BB.SS73.1F	9.1	414	Yes
31	12-17-12	SS.BB24.2F	8.7	257	Yes
32	12-17-12	SS.SS89.1F	9.0	394	Yes
33	2-19-13	BB.BB77.3M	9.1	519	Yes
34	2-19-13	BB.SS71.3M	8.4	284	Yes
35	2-19-13	SS.BB22.4	8.5	273	Yes
36	2-19-13	SS.SS91.1M	9.5	557	Yes
37	2-19-13	BB.BB90.1F	8.3	448	Yes
38	2-19-13	BB.SS73.2F	8.4	318	Yes
39	2-19-13	SS.BB24.3F	8.1	281	Yes
40	2-19-13	SS.SS91.4F	8.5	563	Yes

12-17-12: I was going to try to finish everything up today or tomorrow, but I ran out of reagents. I ordered more but they won't get in for a little while. I'm really frustrated as I was hoping to finish everything up before I head home for the break. Now it looks like it won't get finished until February.

12-18-12: It's good that I didn't extract the other 8 yesterday, as the RIN numbers were all quite low. I'm not sure why, but I need to get that straightened out. I got one of the reagents in the mail today, now I'm just waiting on the other and I will be set to finish everything off.

2-20-13: Just bioanalyzed the last samples. Not great, but D~ says they are good enough - Illumina calls for >8. J~ was fine with it since there are only 8/40 that are between 8 and 9.

*There is one sample that was only 7.4 (BB.SS73.3M) and I re-bioanalyzed it today it went down to 5.6. This says to me that it has some RNase contaminant in it and is being actively degraded. I have some RNA partially extracted and archived that I will finish extracting, but most likely I will need to find a new sample. Though, having 1 out of 40 go bad is fine by me, that's pretty good odds.
*4-1-13 Upon re-extracting this sample the RIN was 8.6 and the concentration was a bit high (so the actual RIN is likely much higher) I can now finish up with the library preps.

RNA extractions and the Bioanalyzer III: the solution

I finally figured it out. Naturally it was one of those mistakes that are considered "dumb" in hindsight, but it sure caused a ton of stress at the time. The problem was that I was not diluting my samples enough when I put them on the bioanalyzer. It's nothing to do with ethanol or chloroform or any of the other variables I spent the last couple months testing. I have been doing RNA extractions just fine the entire time, and then fucking up the quality assay.

Below you can see the differences. On the left there are two samples that I ran on October 1st. The desired concentration is between 200 and 5,000pg/ul and these were at a concentration of 92,448pg/ul and 54,308pg/ul. I diluted them down to the proper concentration and re-ran them yesterday (right column) and they turned out really great (RIN>8 is acceptably good, >9 is perfect).

So, what have I learned? The bioanalyzer is incredibly accurate in it's small range of acceptable concentrations, once you get outside of that range the quality goes way down. The nanodrop is mildly accurate, but can handle a huge range of concentrations. The first step is always to use the nanodrop to get a good idea of the starting concentration of a sample, then dilute the samples to fall within the range of high accuracy of the bioanalyzer. 

There are actually two types of chips for analyzing RNA with the Bioanalyzer: "Nano chips" and "Pico chips." The differences is the concentration they deal well with. Here's a picture:

                                           

Green = Nanodrop

Red = Bioanalyzer Pico chip (20-5000pg/ul)

Blue = Bioanalyzer Nano chip (5-500ng/ul)

It turns out that if I had been using the Nano chips, my RNA probably would have fallen perfectly within the range. That would have saved a bunch of time, heartache, and money, but on the other hand I wouldn't have learned the concentration lesson in such a lasting and exquisitely painful manner.

I now have nano chips which I will be using for my samples and I am going to run the rest of my samples to see whether I ruined them (as I had thought before) or they are actually usable (which it looks like they probably are). Most importantly for my personal self-confidence though, this means that my molecular technique is just fine, it was my understanding of the machines I was using that fell short. A deficit in knowledge is easier to correct and not such a blow to the ego.

RNA extractions and bioanalyzer results II

I have done a couple trials of RNA extractions to figure out what is going wrong (see my last post on rna extractions).

All these were done using liver tissue which had been dissected and snap frozen on dry ice:

I extracted the first two and the results were not great, so I talked with J~ and realized some mistakes (too much tissue, no Beta mercaptoethanol, undergrads using my pipettes...). Then I did the next three rows to correct for those possible errors. I also changed how I'm washing the mortars and pestels: use bleach, then rinse with molecular grade water, use RNase-away and rinse with molecular grade water, then cover with tin foil and dry at 95c, and cool before use.

I won't post the entire bioanalyzer results, but here is a summary of the results:
I also did an experiment where S~ and I each extracted RNA side-by-side to make sure that I wasn't missing a step or doing anything else weird. S~ didn't notice anything, but the results (both hers and mine) were well below 8.

As everything up to this point was only mildly successful if that (none above RIN=8), I decided to do a comparison between completely fresh tissue and that same tissue that had been frozen. I sacrificed an animal and divided the liver into two parts - on went straight on dry ice and the other went directly to the extraction. The one on ice remained there until I finished the first extraction ~1.5hours.
I also realized that I have a second protocol that doesn't require the use of columns at all (it just uses the RNAsolv reagent), so I decided to run these next to each other. I followed the same protocol as above in regard to fresh and frozen tissue. The RNAsolv method ended up with 4 tubes per treatment. I only ran 2 of each on the bioanalyzer.

Results:



The two RNAsolv with fresh tissue didn't work at all and the other two were quite poor. I won't be using that protocol again. As for the Fresh vs Frozen tissue with the kit, it looks like frozen is actually better, though the difference is small. At least I have gotten two 8's though, that's looking up.

Not really sure what to do next. Committee members have said to just keep going until it's right, regardless of how long it takes. But I don't have anything to change, and furthermore I don't feel like I've done anything different even between the earlier 6.6 and this recent 8. The lab tech has said that she didn't realize that she was doing anything different when she suddenly started getting high RINs though so maybe there is still hope for me.

In unrelated news, it turns out that placenta has one of the highest incidences of RNases (along with pancreas). That doesn't explain poor quality liver RNA of course, but is something for me to keep in mind when extracting my placentas in the future...

RNA extractions and bioanalyzer results

I bioanalyzed the "test" RNA placenta sample and it turned out to be slightly degraded: the RNA Integrity Number (RIN) was 7.8 (should be ~10).

        The two tall peaks at 2000 and 4000 are the ribosomal RNA, the small one at ~180 is the marker. The RIN is a measure of quality, essentially it's a ratio between the two ribosomal peaks. The second one (4000) should be much taller than the first, otherwise there has been some degradation that occured and the sample is no good. In this sample, the second is indeed taller than the first, but not enough. I actually think it's calculated by area under the curve rather than height, but close enough. Another sign of bad RNA is the ripples in between the two peaks - it should be fairly smooth, when it's rough, that's a sign of degradation.
       In all, I was kind of expecting this as the sample thawed briefly before I got it in the buffer. As in the last post, I realized this and planned around it for the next time. Then, mistakenly, I assumed that I should go ahead and start on my samples. I extracted 12 (of 40) and bioanalyzed the first 5:

Each sample here has 2 charts, the first is the totalRNA, the second is the miRNA. There will be no ribosomes in the miRNA, so we have no way of gauging quality with those, but as for the others, they were all pretty awful. No RIN was above 7.3 and most didn't have even an estimate. Clearly there is some degradation although that's not the only thing going on - numbers 7 and 9 both have a weird hump early in the read before establishing the baseline and 6 and 8 have a hump near the end. Neither of these things should happen. This also sucks because they were my important precious samples. I do have back ups for them all so it's not a huge deal, but it's scary as I definitely ruined them and I need to not do this again.

The next step is to bioanalyze the other samples that I've extracted already.
After that I need to figure out what is going wrong:
     I will first do another extraction (liver this time - placentas are too valuable) comparing my kit (Omega) with another brand (Qiagen) that we have in the lab and we know works.

1.      If both come out bad then it's my fault and I need to do better with my technique (Yikes, I really hope it's not this one...)
2.      If the Qiagen one is bad and not the Omega, then it's an issue with the kit, and we need to get a new kit (or make new buffers).

Another potential issue is that I used standard alcohol when preparing my buffers not realizing that molecular grade ethanol exists. I will be using molecular grade in the future on of course, but in order to avoid confounding variables in the above experiment I will do everything exactly the same for the Omega extraction as I had before. Afterwards, I will do second test will be to use molecular grade ethanol alongside regular ethanol to determine whether the ethanol is the problem.