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I am trying to isolate total RNA using QIAGEN miRNeasy Mini Kit (50) - Cat #217004 from a 2mm biopsy. While there are only two samples (B4 and DT11) the quality of RNA is bad.

The goal is to use this RNA for RNAseq afterward and qPCR (do the cDNA).

All is done on ice, a centrifuge is cooled down, and no additional DNAse step is added.

What can be the problem? Is this RNA really bad for the goals indicated above?

RNA quality control assay

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    $\begingroup$ What is your problem? Have you measured a A260/280? $\endgroup$
    – Chris
    Jul 21, 2023 at 22:14
  • $\begingroup$ In addition - how were the biopsies stored prior to extraction? You are also using a kit aimed for miRNA - is this what you want or do you want total RNA or mRNA? $\endgroup$
    – bob1
    Jul 21, 2023 at 22:28
  • $\begingroup$ @bob - we aim at mRNA $\endgroup$
    – Lara
    Jul 21, 2023 at 22:31
  • $\begingroup$ @Chris yes, these ratios are 1.97/2.02 correspondingly. The problem is the smear. It is supposed to be 18S + 28S rRNA in general... Or am I wrong? $\endgroup$
    – Lara
    Jul 21, 2023 at 22:34
  • $\begingroup$ @bob biopsies were at -80 $\endgroup$
    – Lara
    Jul 21, 2023 at 22:39

1 Answer 1

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As mentioned in the comments, RIN are about 2.2 - 2.4.

These values are typically too low for RNA-seq, and indicate that the samples are quite highly degraded. One study by Reimann et al1 found that about 35% of the variation in transcript abundance in RNA-seq was caused by RNA degradation. That's not to say you can't sequence these samples, just that you shouldn't rely on the data for transcript abundance measures. The same applies to qPCR - where it is recommended to have RIN above 5.02.

As to what you can do about the RNA being degraded - that all depends on the samples.

It may be that you need to improve storage conditions - store samples (including the biopsies) at or below -80, preferably in the presence of RNase inhibitors. If you can't store the biopsies at -80, put them into and store them in RNA-later or similar as quickly as possible after being taken from the patient.

Extracted RNA should be aliquoted into single-use aliquots, stored at -80, freeze/thawed only once and used in down-stream applications ASAP.

It could also be that you are working in a contaminated environment - you should be using dedicated RNA extraction pipettes, filter tips and good lab practices - certainly don't use pipettes that have been anywhere near PCR products or RNases (cDNA conversion).

Preferably work in a laminar flow cabinet to prevent external contamination of samples and if you can, do RNA work in a room dedicated for that purpose to prevent cross-contamination with DNA from other sources such as PCR products. DNA contamination from PCR is a huge problem - you can get it from aerosols in the air and it WILL show up in your sequencing.

Use tubes from a reliable source that state "RNAse and DNase free" straight out of the bag (for example; don't autoclave the tubes or transfer to another container) and only touch that bag with gloved hands. Wipe work surfaces with RNase-away or similar product before use. Change gloves regularly and make sure you change them if you touch your skin or hair (e.g. you move a strand of hair off your face or scratch an itch - change your gloves!).

It may be that this is just the state of the RNA in these samples. In which case you can only work with what you have. You just need to be very careful about declaring these results when publishing - be very clear on the RIN values and what compensatory analyses you did or didn't do!

Edited to add: if you are new to the field - find some samples you can practise on before working with valuable real samples, which are often a finite resource. This could be cultured cells or samples from a euthanized lab animal (with appropriate ethical approval).

Refs:

  1. Reiman, M., Laan, M., Rull, K. and Sõber, S. (2017), Effects of RNA integrity on transcript quantification by total RNA sequencing of clinically collected human placental samples. The FASEB Journal, 31: 3298-3308. https://doi.org/10.1096/fj.201601031RR

  2. Padhi BK, Singh M, Rosales M, Pelletier G, Cakmak S. A PCR-based quantitative assay for the evaluation of mRNA integrity in rat samples. Biomol Detect Quantif. 2018 Mar 16;15:18-23. doi: 10.1016/j.bdq.2018.02.001. PMID: 29922590; PMCID: PMC6006387.

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