For analysis of miRNA expression, 10 ng of total RNA were reverse transcribed in a total volume of 15 μl using the High Capacity cDNA Reverse Transcription kit and miRNA-specific RT primers (Applied Biosystems). PCR reactions were performed using the TaqMan MicroRNA Assay kit (Applied Biosystems). Each PCR reaction contained 1.33 μl of the RT reaction product, 10 μl of TaqMan 2x Universal PCR Master Mix, and 1 μl of 20x TaqMan MicroRNA Assay reagent in a total volume of 20 μl. Expression of miRNAs was normalized to endogenous snoRNA 202 (assay ID 001232) and/or rat snoRNA (assay ID 001718) expression for each sample using the ∆∆Ct method.
Mirna specific rt primers
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MiRNA-specific RT primers are a set of oligonucleotide sequences designed for the reverse transcription of microRNA (miRNA) molecules. These primers provide a targeted and efficient method for the conversion of miRNA into complementary DNA (cDNA) for further analysis and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Quantstudio 3, by Thermo Fisher Scientific (2 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Purelink mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Powerup sybr green qpcr kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Rneasy plus micro, by Qiagen (1 mentions)
Reverse transcription system, by Promega (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Abi prism 7300 system, by Thermo Fisher Scientific (1 mentions)
Taqman mirna qrt pcr assay, by Thermo Fisher Scientific (1 mentions)
Rnu48, by Thermo Fisher Scientific (1 mentions)
Multiscribe reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
6 protocols using mirna specific rt primers
1
Quantifying miRNA Expression via RT-qPCR
2
Quantification of KSHV miRNA Expression
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Total RNA was extracted using TRIzol (Invitrogen) and treated with DNase I (NEB). cDNA was synthesized with M-MLV Reverse Transcriptase (Promega) using random 9-mers (Integrated DNA Technologies). qPCR was performed using the PowerUP SYBR Green qPCR kit (Applied Biosystems 100029284) using appropriate primers on a QuantStudio 3 (Thermo Scientific).
For quantitation of KSHV-pri-miRNAs, RNA was isolated using the PureLink miRNA isolation kit (Invitrogen K1570-01). 1 ng of RNA was used for cDNA synthesis using TaqMan MicroRNA reverse transcription kit (Applied Biosystems 4366596) with miRNA specific RT primers (Applied Biosystems).
The mature KSHV miRNA were quantified using 0.67 μl of cDNA and microRNA primer probe sets (Applied Biosystems) (see Supplementary TableS2 for details), with TaqMan Universal PCR Master Mix, no AmpErase UNG (Applied Biosystems 4324018) according to manufacturer’s instructions.
For quantitation of KSHV-pri-miRNAs, RNA was isolated using the PureLink miRNA isolation kit (Invitrogen K1570-01). 1 ng of RNA was used for cDNA synthesis using TaqMan MicroRNA reverse transcription kit (Applied Biosystems 4366596) with miRNA specific RT primers (Applied Biosystems).
The mature KSHV miRNA were quantified using 0.67 μl of cDNA and microRNA primer probe sets (Applied Biosystems) (see Supplementary Table
3
Endothelial Cell RNA Isolation and RT-PCR
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Prior to reverse transcription polymerase chain reaction (RT-PCR), total RNA from endothelial cell cultures was isolated using Total RNA Isolation System RNeasyPlus Micro (Qiagen, Germany), following manufacturer's instructions. The purity and amount of RNA were determined by measuring the OD at a ratio of 260 to 280 nm. One microgram of total RNA was transcribed into cDNA by Reverse Transcription System (Promega, Madison, WI, USA). For microRNA measurements, 5 ng of total RNA was reverse-transcribed using the TaqMan®MicroRNA Reverse Transcription Kit and miRNA-specific RT primers (Applied Biosystems).
4
Quantitative Expression Analysis of miR-128
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Expression of miR-128 was evaluated using qRT-PCR. Briefly, total RNA was extracted from tissues or cells using RNeasy kits (QIAGEN, Valencia, CA, USA). RNA was reversely transcribed into cDNA using miRNA-specific RT primers (Thermo Fisher Scientific Inc., Waltham, MA, USA). The used primer sequences (Invitrogen, Shanghai, China) were as follows: miR-128, 5′-TCCGATCACAGTGAACCGGT-3′ (forward) and 5′-GTGCAGGGTCCGAGGT-3′ (reverse); U6, 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse). qRT-PCR was performed by a TaqMan miRNA qRT-PCR assays (Applied Biosystem, Waltham, MA, USA) with ABI-Prism 7300 System (Applied Biosystem, Waltham, MA, USA). Expression of miR-1258 was measured using 2−ΔΔCt as described previously [27 (link)].
5
Quantitative miRNA Expression Analysis in Tumor Tissues
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miRNA-specific expression assays were carried out on fixed mass RNA inputs of 250 ng. Total cellular RNAs were reverse transcribed using miRNA-specific RT primers (Thermo Fisher) in the presence of MultiScribe Reverse Transcriptase (Thermo Fisher). Resulting cDNAs were then amplified in miRNA-specific TaqMan fluorescence assays (Thermo Fisher). For miRNA assays, the standard, well-validated endogenous RNA control RNU48 (Thermo Fisher) was used for normalizing. miRNA expression (Ct) was normalized (ΔCt) and tumor miRNA expression compared with benign tissue expression via the standard ΔΔCt method19 (link),20 (link) where ΔΔCt=ΔCttumor–ΔCtcontrol and fold change is 2−ΔΔCt. Statistical significance was assessed by a two-tailed t-test with unequal variance.21 A P-value<0.05 was taken as statistically significant.
miRNA expression correlations were carried out pairwise across all five unique members of the miR-503 cluster on all 24 tissues in the sample using Pearson product–moment correlation. Normalized expression values (ΔCt) were the input data. Statistical significance was assessed using a standard look-up table with n–2 degrees of freedom. A P-value<0.05 was taken as statistically significant.
miRNA expression correlations were carried out pairwise across all five unique members of the miR-503 cluster on all 24 tissues in the sample using Pearson product–moment correlation. Normalized expression values (ΔCt) were the input data. Statistical significance was assessed using a standard look-up table with n–2 degrees of freedom. A P-value<0.05 was taken as statistically significant.
6
Quantifying miRNA and mRNA Expression
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Total cellular RNA was isolated from cultured cells using miRNeasy mini kit (QIAGEN), according to the manufacturer’s protocol, and stored at −80°C in RNase-free water. Reverse transcription was performed on 50 ng total RNA using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) and olidgo-d(T) or miRNA-specific RT primers (Thermo Fisher Scientific). qPCR was performed using TaqMan MicroRNA Assays (Thermo Fisher Scientific) for 40 cycles on a QuantStudio 3 (Applied Biosystems). Relative expression of miRNAs and mRNA was calculated in comparison to the small nucleolar RNA (snoRNA) U18 or endogenous GAPDH, respectively, using the 2−ΔCt method.
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