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Protocol recommendation for RNA or cDNA clean up #1834

Closed valeste closed 6 months ago

valeste commented 6 months ago

Experiencing issues getting expected relative standard curves and acceptable E values for all of my genes of interest. I suspect issue to be related to the quality of the cDNA (inhibitors in the qPCR reaction mix?).

Protocol leading to cDNA synth:

  1. RNA extracted using Qiagen RNeasy Mini Kit with 10% β-mercaptoethanol (β-ME) in RLT buffer and QiaShredder column step supplemented to protocol.

  2. Quantify RNA with NanoDrop.

  3. gDNA elimination with Invitrogen TURBO DNA-free Kit (Cat. No. AM1907).

  4. Requantification of gDNA eliminated RNA with NanoDrop:

Date Sample ID RNA Concentration (ng/uL) A260/A280 260(10mm)
09/30/2023 9302023 0-R1 pilot 19.2 1.55 0.48
09/30/2023 9302023 0-R2 pilot 113.2 1.85 2.83
09/30/2023 9302023 0-R3 pilot 152.4 1.87 3.809
09/30/2023 9302023 0-R4 pilot 67.1 1.77 1.676
09/30/2023 9302023 5-R1 pilot 22.4 1.66 0.56
09/30/2023 9302023 5-R2 pilot 30.6 1.68 0.764
09/30/2023 9302023 5-R3 pilot 29 1.67 0.726
09/30/2023 9302023 5-R4 pilot 45.1 1.77 0.128
09/30/2023 9302023 45-R2 pilot 87.6 1.82 2.14
09/30/2023 9302023 45-R3 pilot 13.8 1.47
09/30/2023 9302023 45-R4 pilot 84.8 1.83 2.12
10/25/2023 10252023 0-R1 E1 12.1 1.55 0.305
10/25/2023 10252023 0-R2 E1 45.9 1.83 1.148
10/25/2023 10252023 0-R3 E1 78.4 1.85 1.959
10/25/2023 10252023 0-R4 E1 38.5 1.8 0.962
10/25/2023 10252023 5-R1 E1 83.3 1.82 2.082
10/25/2023 10252023 5-R3 E1 27.1 1.69 0.676
10/25/2023 10252023 5-R4 E1 69.5 1.84 1.737
10/25/2023 10252023 45-R1 E1 50 1.78 1.249
10/25/2023 10252023 45-R2 E1 57.3 1.84 1.433
10/25/2023 10252023 45-R3 E1 43.5 1.73 1.087
10/25/2023 10252023 45-R4 E1 60.1 1.83 1.505
11/15/2023 11152023 0-R1 E2 86.5 1.86 2.163
11/15/2023 11152023 0-R2 E2 54.7 1.66 1.367
11/15/2023 11152023 0-R3 E2 37.4 1.72 0.935
11/15/2023 11152023 5-R1 E2 58.4 1.66 1.459
11/15/2023 11152023 5-R2 E2 69.7 1.69 1.743
11/15/2023 11152023 5-R3 E2 33.7 1.51 0.843
11/15/2023 11152023 45-R2 E2 34.5 1.44 0.863
11/15/2023 11152023 45-R3 E2 16.2 1.5 0.405
11/29/2023 11292023 0-R1 E3 72.4 1.78 1.809
11/29/2023 11292023 0-R2 E3 60.3 1.78 1.507
11/29/2023 11292023 0-R3 E3 21.4 1.61 0.534
11/29/2023 11292023 5-R1 E3 12.7 1.43 0.318
11/29/2023 11292023 5-R2 E3 15.5 1.57 0.387
11/29/2023 11292023 5-R3 E3 64.4 1.76 1.609
11/29/2023 11292023 45-R1 E3 80.6 1.82 2.016
11/29/2023 11292023 45-R2 E3 49.3 1.77 1.231
11/29/2023 11292023 45-R3 E3 64.3 1.79 1.607

Drop in RNA 260/280 of about 0.2 across each sample

Refer to notebook post for full details if needed.

Is it normal for the quality of the RNA to drop so much after using Turbo DNA free kit?

github-actions[bot] commented 6 months ago

Thanks for posting an issue!

kubu4 commented 6 months ago

Okay, I'll start this with a "warning" - there's a LOT to unpack here, because you're not really just asking about the RNA.

You're real concern is the qPCR results.

So, let's get into it! Be prepared for a lot of reading and a lot of questions!

In an attempt to make this a bit easier, I'll try to divide it into sections.

RNA

Is it normal for the quality of the RNA to drop so much after using Turbo DNA free kit?

Yes, this is not surprising. You're enzymatically fragmenting DNA. Enzymes are proteins. The 260/280 ratio measures absorbance of nucleotides (~260nm) relative to the absorbance of protein (~280nm). Thus, you've added more protein to your samples, so your sample "purity" is expected to decrease.

Questions:

  1. Do you have access to a Qubit (the Roberts Lab has one, if you don't already)? The Qubit is far more sensitive and accurate than the NanoDrop, but will not provide you with A260/280 and A260/230 ratios.
  2. Do you have the A260/230 ratios? This information will provide a clearer picture on whether or not there are any possible inhibitors that got carried over (things like phenol, alcohol, excessive salts result in a poor A260/230 ratio).

DNase

1a. Which DNase protocol did you follow - standard or rigorous? 1b. What was the makeup of your DNase reactions (e.g. 1uL DNase, 5ug of RNA, 25uL water, etc)?

cDNA

  1. How much RNA did you use for your reverse transcription reactions?
  2. What was the makeup of your reverse transcription reactions (e.g. 5uL buffer, 5ug RNA, 10uL, 1uL dNTPs)?

PCR and Primers

Your two gels mostly appear to consist of primer dimers (except ACTb).

Questions:

  1. What are your expected amplicon sizes for each primer set? I did see a note in your notebook entry on primer design that said you were targeting 500bp, but it'd be helpful/useful to include that info in your table(s).
  2. What was the makeup of your end-point PCR reactions (e.g. 10uL 2x polymerase, 0.5uL 10uM Pf, 0.5uL Pr, 9uL H2O, etc)?
  3. What were your cycling parameters for end-point PCR?
  4. Where is your 18s primer set on either of the two gels?
  5. What were your cycling parameters for your qPCR reactions?
  6. Were your NTCs clean? It be cool if you could color code your amplification plots to have the NTCs be a different color than the rest of the samples - that would make it easier to quickly evaluate this aspect of things.

Okay, I think that's it for now. If you're so inclined, it could be good to go over this stuff at lab meeting, as there are certainly others in the lab who may have to do qPCR and it would be a good excuse to discuss all the ins/outs. Plus, sometimes a topic with this much breadth can be a bit difficult to handle via back-and-forth on GitHub.

However, the decision is yours! I'll happily help out here or during lab meeting or even a one-on-one video chat if desired.

We'll get things figured out!

sr320 commented 6 months ago

Agree with @kubu4 - Science hour would have loved this, but otherwise lets tackle at lab meeting. To offer crude / correct? framework to how I would consider qPCR output.

  1. NTC amp - start over.
  2. Melt curve single peak - next step. Not- try one more PCR with higher anneal - and/or then move to new primer.
  3. Amplification looks good? Done.

(I could be corrected but no need for standard curves) - but related if I am interpreting correct - technical replicates concern me.

@valeste it would be good to answer questions @kubu4 posed here /and or notebook before we chat.

valeste commented 6 months ago

RNA

  1. Do you have access to a Qubit (the Roberts Lab has one, if you don't already)? The Qubit is far more sensitive and accurate than the NanoDrop, but will not provide you with A260/280 and A260/230 ratios.

No, I don't currently have access to a Quibit out at UW Tacoma.

  1. Do you have the A260/230 ratios? This information will provide a clearer picture on whether or not there are any possible inhibitors that got carried over (things like phenol, alcohol, excessive salts result in a poor A260/230 ratio).

The NanoDrop available for use at UW Tacoma is a NanoDrop Lite and only reports 260/280 ratios. I agree it would be good to have info for the 260/230 ratios as well.

DNase

1a. Which DNase protocol did you follow - standard or rigorous?

Standard

1b. What was the makeup of your DNase reactions (e.g. 1uL DNase, 5ug of RNA, 25uL water, etc)?

gDNA Elimination Component per 1 rxn (uL)
10x DNase Buffer 2
Turbo Enzyme 1
RNA sample 20
Total 23

cDNA

  1. How much RNA did you use for your reverse transcription reactions?

96.8 ng per sample rxn

  1. What was the makeup of your reverse transcription reactions (e.g. 5uL buffer, 5ug RNA, 10uL, 1uL dNTPs)?
cDNA synthesis Component per 1 rxn (uL)
SuperScript IV VILO Master Mix 2
Nuclease-free water variable
RNA sample variable
Total 8

qPCR

1.What are your expected amplicon sizes for each primer set? I did see a note in your notebook entry on primer design that said you were targeting 500bp, but it'd be helpful/useful to include that info in your table(s).

Below is the info for amplicon length per primer set. I couldn't find all the info for primers pulled from the literature but aside from ACTb all are below 200 bp as I described for my aims in designing.

Number Gene Forward Primer (5'-3') Reverse Primer (5'-3') NCBI Accession Number (If complete CDS) Botryllus Contig Sequence (if no complete CDS was found) Amplicon Length Source
1 BAX CATTGACCTTGCGTGTTATTGG AAGATGTCCTTGGTGGGTTG KU948200.1 None 97 in house
2 POU3 CAGTAGGTCTTCGCATCCATC CGTTTGCATCGTTGGTGTAAG KU948203.1 None 80 in house
3 PolB GGCTTCACAATCAATGAGTATTCC AGCAATCTTTCCGCATTCATTC None botctg092056 NA in house
4 TNF GTTTCAGTCAACACCTTGTGTC AATCTCGACCGCTGACTTATAC None botctg065850 131 in house
5 SOXB1 GGGAATGTGTTCATCCTCTGG TGAATCCAGGAGTTACTCAAGTTC KU948203.1 None 116 in house
6 MYC GAAGTCCTGGCTAAGATGCTAC AGCGTCAAGAAGGAAGTACG KU948203.1 None 119 in house
7 AIF1 GGATTCTCTCACCGATGGAATAG CCTGACTTGGAGCCTTTATGG KU948202.1 None 102 in house
8 SOD1 CAATCCATATAACAAGGTTCATGGC TGATATCCACACAGGCACAAC EE743567.1 None 107 in house
9 PARP1 AATTTGGCACCGCTCATTTC TGCAAGACCTTCAGCTTCTC KU948201.1 None 94 in house
10 SIRT6 ATTAGTACCGCGTCTGGAATAC GGATGGAATCGCTTCTTCAAATC None botctg109566 114 in house
11 IAP7 GGTCTCTTCACGGACATCTTC GCTCAGTATGTTGTGCTTGTTC KU948203.1 None 111 in house
12 HSP70 CGAGAAGTTGAAGGACAAGATCTCCG TCTCTGCAGTCTGGTTGTTGTCGAGC NA NA NA doi:10.1016/j.ydbio.2017.10.023
13 Mortalin TTCATGATACCGAAACCAAGATGGACG AATTTCCTCCTTGATGGCATCCACC KY212120.1 None 79 doi:10.1016/j.ydbio.2017.10.023
14 ACTb GTAGGTAGTCTCGTGAATTC CACGCCATCTTGCGTCTGGA NA NA 321 doi:10.1016/j.dci.2006.12.009
15 18s GGCAGCCTCCGGGAAACCAAAGTC CTGGTGGTGCCCTTCCGTCAATTC NA NA NA doi:10.1016/j.ydbio.2017.10.023

What was the makeup of your end-point PCR reactions (e.g. 10uL 2x polymerase, 0.5uL 10uM Pf, 0.5uL Pr, 9uL H2O, etc)?

EP-PCR Component per 1 rxn (uL)
PCR Master Mix (brand TBD) 12.5
Nuclease-free water 9.5
Forward Primer (5 uM) 1
Reverse Primer (5 uM) 1
1:3 dilution of pooled (per treatement) cDNA 1
Total 10

What were your cycling parameters for end-point PCR?

Need to double check but roughly these parameters.

Stage Step Temperature (C) Duration (sec) Cycles
PCR Denature 95 15 40
PCR Anneal/Extend 60 60 40
Hold Hold 4 infinity 1

Where is your 18s primer set on either of the two gels?

Gel annotation is mislabeled. Where it says 40s it should say 18s.

What were your cycling parameters for your qPCR reactions?

Stage Step Temperature (C) Duration (sec) Cycles
Hold Hold 50 120 1
Hold Hold 95 600 1
PCR Denature 95 15 40
PCR Anneal/Extend 60 60 40
Melt Curve Denature 95 15 1
Melt Curve Anneal/Extend 60 60 1
Melt Curve Dissociation 95 1 1

Were your NTCs clean? It be cool if you could color code your amplification plots to have the NTCs be a different color than the rest of the samples - that would make it easier to quickly evaluate this aspect of things.

The only NTCs that were "clean" were the ones for the primers I labeled as trash that failed to amplify in target reactions with cDNA. Beta actin also had a clean NTC.

Note that for some of the GOI's like AIF1 and SOD1, the NTC Ct value is nearly identical to the rest of the Ct values. (Meaning the Ct value remains the same across decreasing concentrations of cDNA and in the NTC, likely a primer dimer artifcat and no amplification potentially occuring?)

Target Name Task CT
BActin NTC Undetermined
BAX NTC 32.548
SOD1 NTC 26.921
SIRT6 NTC Undetermined
IAP7 NTC 32.048
HSP70 NTC Undetermined
Mortalin NTC 32.660
18s NTC 32.623
POU3 NTC 33.793
PolB NTC Undetermined
TNF NTC Undetermined
MYC NTC 32.153
AIF1 NTC 27.873
PARP1 NTC Undetermined
sr320 commented 6 months ago

Note that for some of the GOI's like AIF1 and SOD1, the NTC Ct value is nearly identical to the rest of the Ct values. (Meaning the Ct value remains the same across decreasing concentrations of cDNA and in the NTC, likely a primer dimer artifcat and no amplification potentially occuring?)

this is liking simply contamination.

sr320 commented 6 months ago

Also, have a look at https://robertslab.github.io/resources/Lab-Protocols/#qpcr-data-analysis

kubu4 commented 6 months ago

Thanks for all this! Here's another lengthy response!

DNase

Reverse Transcription

End-point PCR

qPCRs

So, where does that leave us?

Well, personally, I'd run each primer set again (in triplicate, including NTCs), using just your undiluted pooled cDNA - don't bother with standard curves. Need to make sure qPCR reactions for each primer set are working (or not) before worrying about primer efficiencies. Try to improve technical reps and clean NTCs and then make further decisions.