Hello Everyone,

I’m testing the expression of 2 miRNAs (I will call then miR-1 and miR-2) from a fly, using the NCode™ miRNA First-Strand cDNA Synthesis Kit (Life Technologies) protocol in order to do the cDNA, and SYBR® Green PCR Master Mix (Life Technologies) to perform the qPCR. I choose these miRNAs because they appear to be high expressed in my samples, due the reads count of NGS (Illumina) results. But, I’m having problems with my Cts values, and I can’t think those values are real. Bellow I will put some more details:

My Procedures:

I) Total RNA was isolated using Trizol Reagent. The quality of the extractions were verify by electrophoresis (1ug of RNA in 1% denaturante Agarose Gel with Gel red, no smear and RNA seems to be ok!), Nanodrop (The ratio 260/280 is between 1.9 – 2.1 as expected for pure RNA) and the quantifications were done in Qubit (Between 900 ng to 1 ug per ul could be recovered, what’s nice).

II) I’m using Ncode kit (the described previously) for cDNA synthesis. The starting RNA is 2.5 ug (the maximum allowed for the kit). First for the poly-A Tailing (final volume of 25ul) I’m incubating the reaction at 37ºC for 15 min. Then I’m using 4ul of this reaction to make the first strand cDNA, with Universal RT primer provide and SuperScript III RT (50°C for 50 min and then 85ºC for 5 min).

III) Then I dilute the cDNA 10 fold (1:10) and apply 2ul in a 20ul qPCR reaction using SYBR® Green PCR Master Mix (10ul SYBR, 0.4 ul of each primer (10uM) and 7.2ul of DEPC water). The direct primers used for the miRNAs are identical to the mature miRNAs that I will analyze, (miR-1 primer Tm is 62ºC and miR-2 primer Tm is 60ºC) and the reverse one (provide with Ncode) is complementary to the Poly-A tail added. My control is U6, and I’m using a set of primer that works well at 60°-65°C.

IV) The amplifications are done in Applied Biosystem 7500, by: 50°C for 2 min, 95°C for 2 min., followed by 40x 95ºC for 15 seg and 60ºC for 30 seg. The Dissociations curves are been also made. All reactions are done in triplicates, RT- and NTC- controls.

My results so far:

1. The Cts values for U6 are around 23 or 24. What’s very nice when compared with my previous results. The Melting curve also is perfect!! Just one amplification at about Tm 77°C, and the peak of the derivative curve around 0.240 to 0.280.

2. But….the Cts Values for miR-1 and miR-2 are about 33, and again, it’s not seems to be real. The Melting curve for miR-1 appear with two distinct peaks (I do not try yet increase the annealing temperature for this miRNA) and the Melting curve for miR-2 appears with a single peak. However, the peak of the derivative curve for miR-1 is around 0.07 and for miR-2 around 0.035, what’s very low.

3. After the qPCR reaction I run a 4% agarose gel and I could see the amplifications about 50bp, what is expected. Of course the U6 amplification bands were stronger than amplifications of the miRNAs, but both were present.

What I already tested

1° The amount of RNA used for cDNA reaction. I started with about 700ng, and my Cts values for U6 were about 33 – 34. After started using 2.5 ug the Cts lowered, but not for the miRNAs.

2° I also try isolated (enrichment) miRNAs from total RNA using mirVana™ miRNA Isolation Kit and then use 2.5ug of small RNA enrichment to performed cDNA. It didn’t work; I keep getting the same results for U6 and my miRNAs.

3° I already try using 2ul of undiluted cDNA and 1:5 diluted cDNA, but apparently no significant results were obtained (compared with previously one).

My next steps

1. I saw another kit that makes the same steps of Ncode, the qScript (Quanta biosciences). One difference is that the poly-A Tailing is performed at 37°C for 60 min, when using more than 100ng of RNA, instead of 15 min (Ncode). After that the reaction is maintained at 70°C for 5 min (I believe for inactivate the Poly-A polymerase). So two things I’m thinking (1°) the Poly-A tailing that I’m performing is not efficient enough to 2.5ug of total RNA that I’m using, and (2°) maybe some reagents of this step are hindering my next steps. Perhaps it’s better performed the poly-A Tailing with Ncode kit as suggested by qScript.

2. Once 2.5ug of starting RNA, instead of 800ng, increase the efficient of cDNA reaction, maybe using about 5ug can improve it further. The Only problem is that Ncode (as far as I know) only accept a maximum of 2.5ug of RNA for cDNA reaction.

So, someone had a similar problem? Do my next moves make sense or is there a better test or other ideas?

Thank you so much,

Daniel.