Expression of miR-17, miR-21, miR-18a and let-7g in B16 cells was analyzed using stem-loop PCR technology43 ,44 (link). cDNA synthesis was carried out using SuperScript III reverse transcriptase (SSIII RT, Invitrogen, USA) as previously described45 (link). The RT and PCR primers used in the study are presented in Table S1 (Supplementary information). PCR amplification was carried out in a total volume of 20 µl, using Maxima Hot Start Taq DNA polymerase (Thermo Scientific, USA), 1 × PCR Buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 1 × EvaGreen (Biotium, Hayward, USA), and 0.2 mM of PCR sense and antisense primers. The reaction was performed with initial preheating at 94 °C for 4 min and 40 cycles of 94 °C for 40 s, 61 °C for 30 s, and 72 °C for 30 s, followed by melting point determination. The obtained PCR data were analyzed using standard Bio-Rad iQ5 v.2.0 software. For each sample, the threshold cycle (Ct) was determined. Quantitative assessment of the level of transcripts representation and relative miRNA expression was performed by comparing the Ct values for miR-17 and a reference U6.
Iq5 v 2
Manufactured by Bio-Rad
The IQ5 v.2.0 software is a real-time PCR data analysis software developed by Bio-Rad. It provides users with the tools to analyze and interpret data generated from real-time PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Evagreen, by Biotium (2 mentions)
Maxima hot start taq dna polymerase, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Iq5 multicolor real time pcr detection system, by Bio-Rad (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Iq5 cycler, by Bio-Rad (1 mentions)
Biomaster hs qpcr sybr blue master mix, by Biolabmix (1 mentions)
Sybr green 1 fluorescent dye, by Biolabmix (1 mentions)
Biomaster hs qpcr sybr blue mix, by Biolabmix (1 mentions)
Iq sybr green master mix, by Bio-Rad (1 mentions)
Myiq thermal cycler, by Bio-Rad (1 mentions)
6 protocols using iq5 v 2
1
Quantitative Analysis of miRNA Expression
2
Quantitative PCR analysis of Luc gene
3
Quantification of MDR1 Gene Expression
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Total RNA from leukemic cells was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA quantification was performed with SYBR Green-based real-time qPCR using IQ5 Cycler (Bio-Rad, Hercules, CA, USA). The cDNA was amplified in a total volume of 20 µL containing 2 µL of cDNA template, RNA specific primers (1 µM), and BioMaster HS-qPCR SYBR Blue master mix with SYBR Green I fluorescent dye (Biolabmix, Novosibirsk, Russia). The following primers were used: MDR1_F: 5’-AGAGAATCCCCTCCAGATAAGA–3’ and MDR1_R: 5’-AAGCCTATTCCATTTTGAACTTTCT-3’, GAPDH_F: 5’-GTGAAGGTCGGAGTCAAC-3’ and GAPDH_R: 5’-TGGAATTTGCCATGGGTG-3’. All measurements were done in triplicate. The amount of RNA was calculated from the number of cycles using standard curves and the results were normalized to glyceraldehyde 3 phosphate dehydrogenase (GAPDH). The obtained PCR data were analyzed using Bio-Rad iQ5 v.2.0 software.
4
Quantifying miRNA and mRNA Expression in B16 and RLS40 Cells
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After in vitro transfection of B16 and RLS40 cells as described in Section 2.2 , total RNA was extracted from tumor cells at time points of 24 and 72 h using TRIzol Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. The level of miRNAs was measured using stem-loop qPCR technology [37 ,38 (link)]. cDNA synthesis was carried out using MuML-V reverse transcriptase (Biolabmix, Novosibirsk, Russia) according to the manufacturer’s protocol. The RT and PCR primers used in the study are listed in Table S1 . PCR amplification was carried out using BioMaster HS-qPCR SYBR Blue mix (Biolabmix, Novosibirsk, Russia) according to the manufacturer’s protocol. The obtained qPCR data were analyzed by standard Bio-Rad iQ5 v.2.0 software. For each sample, the threshold cycle (Ct) was determined. Quantitative assessment of the level of transcript representation, relative miRNA and mRNA expression was performed by comparing the Ct values for miRNA and mRNA with HPRT1 and GAPDH mRNAs used as references.
5
qRT-PCR Primer Design and Validation
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Primer3 (http://frodo.wi.mit.edu/ ) was used to design amplicons for qRT-PCR experiments using a primer size of 23 bp, a melting temperature of 62°, and designed to amplify a region of 60−100 bps. When possible, the amplicon, or the primers themselves, was designed to span splice junctions to minimize the amplification of contaminating genomic DNA. Primer pairs were tested for amplification efficiency with a wild-type cDNA dilution series. The following primers were used: Gadd45 (1: qGadd45_F1 and qGadd45_R1; 2: qGadd45_F2 and qGadd45_R2); RP49: qRP49_F and qRP49_R; Copia; qCopia_F and qCopia_R; and tra : qtra_F and qtra_R. All qRT-PCR analysis was conducted using iQ SYBR Green master mix and a MyiQ thermal cycler running iQ5 v2.0 software (Bio-Rad, Hercules, CA). RP49 (RpL32 ) served as a reference gene in all cases. Primer sequences listed in Table S4 .
6
Stem-loop RT-qPCR for miRNA Expression
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Expression of miRNA in RLS 40 cells was analyzed using stem-loop PCR technology [66, 67] . cDNA synthesis was carried out using SuperScript III reverse transcriptase (SSIII RT, Invitrogen, USA) as previously described [68] . The RT and PCR primers used in the study are presented in Table S1, "Supplementary data". PCR amplification was carried out in a total volume of 20 µl using Maxima Hot Start Taq DNA polymerase (Thermo Scientific, USA), 1×PCR Buffer, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 1×EvaGreen (Biotium, Hayward, USA), and 0.2 mM of PCR sense and antisense primers. The reaction was performed with initial preheating at 94°C for 4 min and 40 cycles of 94°C for 40 s, 60°C for 30 s, 72°C for 30 s, followed by a melting point determination. The obtained PCR data were analyzed by standard Bio-Rad iQ5 v.2.0 software. For each sample, the threshold cycle (Ct) was determined. Quantitative assessment of the level of transcripts representation and relative miRNA expression was performed by comparing the Ct values for miRNA and references U6 and Rpl30.
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