Purified miRNA was utilized for cDNA synthesis using the qScript miRNA cDNA Synthesis Kit (Quantabio) according to manufacturer’s instructions (Quantabio, 2020). Briefly, 2 µl of Poly(A) Tailing Buffer (5X), 1 µl of Poly(A) Polymerase, and 7 µl of 500 ng/μl purified miRNA solution were added to a PCR tube to give a final volume of 10 µl. The resulting mixture was then incubated in the C1000 thermal cycler (Bio-Rad) at 37°C for 60 min followed by incubation at 70°C for 5 min. Once finished incubating, 9 µl of miRNA cDNA Reaction Mix and 1 µl of qScript Reverse Transcriptase were added to the PCR tube to give a final volume of 20 µl. The resulting mixture was then placed back in the Thermal Cycler at 42°C for 20 min, followed by incubation at 85°C for 5 min. The resulting cDNA was stored at −20°C prior to qRT-PCR analyses.
Qscript mirna cdna synthesis kit
Manufactured by Quantabio
Sourced in United States
The QScript miRNA cDNA Synthesis Kit is a laboratory equipment product designed for the reverse transcription of mature microRNA (miRNA) molecules into complementary DNA (cDNA). The kit provides the necessary reagents and protocols to efficiently convert miRNA into cDNA, which can then be used for further analysis or downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
Mirneasy serum plasma advanced kit, by Qiagen (1 mentions)
Faststart universal sybr green master, by Roche (1 mentions)
Sybr green, by Takara Bio (1 mentions)
Perfecta sybr green supermix low rox, by Quantabio (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Direct zol rna isolation kit, by Zymo Research (1 mentions)
Perfecta preamp supermix, by Quantabio (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Rotor gene 3000, by Qiagen (1 mentions)
9 protocols using qscript mirna cdna synthesis kit
1
miRNA cDNA Synthesis and Storage
2
Serum miRNA Extraction and Quantification
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Serum miRNA was extracted using the recommended spike-in control cel-miR-39-3p approach (miRNeasy Serum/Plasma Advanced kit; QIAGEN). MiRNAs were reverse transcribed with the qScript miRNA cDNA synthesis kit (QuantaBio, Beverly, MA, USA). Gene expression of let-7e-5p was measured by qPCR in duplicates using the FastStart Universal SYBR Green Master (Roche, Basel, Switzerland), self-designed qPCR primers (ESM Table 2 ) and a universal primer from the qScript kit. Expression was normalised to the spike-in control cel-miR-39-3p expression using the ΔΔCt method (see ESM Methods ).
3
Quantitative RT-PCR Analysis of RNA Transcripts
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Total RNA was extracted using TRIzol reagent (Biosntech, Beijing, China). RNA quality was analyzed using a NanoDrop 1000 (Thermo Fisher Scientific, Inc.). SYBR Green (Takara Biotechnology, Co., Ltd., Dalian, China) was used for qRT-PCR. Amplification was performed using an ABI 7500 real-time PCR system. A qScript miRNA cDNA synthesis kit (Quantabio, Beverly, CA, USA) was used for cDNA synthesis. Gene expression levels were analyzed using the 2−△△CT method. The internal controls for the expression levels of miRNAs and lncRNAs were U6 and GAPDH, respectively. The primer sequences used were as follows.
LncRNA MINCR, 5′-CTAATTCACCTGGCCCGAG-3′ (forward)
5′-CGGCTAGAATCCCAAGG-3′ (reverse);
miR-146b-5p, 5′-CTGAATGTGAAGAGGATGT-3′ (forward)
5′-GTTCTTCGGCCTCCGGGCCC-3′ (reverse);
miR negative control (miR-NC), 5′-UUCUCCGAACGUGUCACGUTT-3′ (forward)
5′-UGAGAACUGAAUUCCAUAGGC UG-3′ (reverse);
IL-1β, 5′-CTTCAGGCAGCCAGTATCAATC-3′ (forward)
5′-TGCAGTGGTCTTATGGGAACGT-3′ (reverse);
MCP1, 5′-ACAACCAAGGCCTTCCTTAC-3′ (forward)
5′-TCTCATTCCCACGATTCCCCAG-3′ (reverse);
TNF-α, 5′-CGAGTGGCAAGCCTGGAGGCC-3′ (forward)
5′-GTCTTGGAGATCCATCCGGTTG-3′ (reverse);
GAPDH, 5′-AACGAACCTTTCATTGAC-3′ (forward)
5′-CACCACTCTTACAGCAACT-3′ (reverse).
4
Circulating miRNA Profiling for Metabolic Disorders
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A panel of 16 miRNAs was selected (Supplementary Table 1 ) due to published association with lipid and glucose metabolism and related disorders [23 (link)–25 (link), 30 (link), 32 (link)]. Circulating miRNA expression was analyzed after purification of RNA from serum using the miRNeasy Kit (Qiagen, Hilden, Germany). cDNA was synthesized with qScript miRNA cDNA Synthesis Kit (Quantabio, Beverly, MA, USA). miRNA quantification was completed by using PerfeCTa SYBRGreen SuperMix Low ROX (Quantabio) in real-time qRT-PCR. Supplementary to the commercially available kits, oligonucleotides for selected miRNAs (Supplementary Table 1 ) were purchased from Eurofins. Obtained CT values of miRs of interest were multiplied with −1 and normalized to the mean of CT of miRNAs 16, 24, 25, and 26 that served as internal controls [42 (link), 43 (link)]. Obtained −ΔCT values give the logarithmic relative expression.
5
Quantitative Analysis of miRNA and mRNA Expression
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Total RNA was extracted from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using the qScript miRNA cDNA Synthesis kit (Quantabio), according to the manufacturer's protocol. qPCR was subsequently performed using a TaqMan™ Real Time PCR Mix (Thermo Fisher Scientific, Inc.). The following primer pairs were used for qPCR: GAPDH forward, 5′-AATGGATTTGGACGCATTGGT-3′ and reverse, 5′-TTTGCACTGGTACGTGTTGAT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; Neurod4 forward, 5′-AGCTGGTCAACACACAATCCT-3′ and reverse, 5′-TTCCATAAGAGCCCGGTCTTC-3′; and miR-340-5p forward, 5′-GCGGTTATAAAGCAATGAGA-3′ and reverse, 5′-GTGCGTGTCGTGGAGTCG-3′. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 8 min; followed by 40 cycles at 95°C for 15 sec and 60°C for 30 sec; and a final extension at 70°C for 35 sec. Expression levels were quantified using the 2−ΔΔCq method (26 (link)) and normalized to GAPDH or U6.
6
Saliva RNA Extraction and miRNA Profiling
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Whole saliva was collected from subjects in a sterile 50 ml tube and 500
microliters was aliquoted and mixed 1.5 ml of TRIzol LS reagent (Life
Technologies). RNA was isolated using the Direct-zol RNA isolation kit (Zymo
Research) per manufacturer protocol and as previously reported.9 (link) Seven µl of purified RNA was reverse transcribed using the qScript miRNA
cDNA synthesis kit (Quanta bio). Expression of miRNAs was analyzed by real time
PCR as described previously.9 (link)
microliters was aliquoted and mixed 1.5 ml of TRIzol LS reagent (Life
Technologies). RNA was isolated using the Direct-zol RNA isolation kit (Zymo
Research) per manufacturer protocol and as previously reported.9 (link) Seven µl of purified RNA was reverse transcribed using the qScript miRNA
cDNA synthesis kit (Quanta bio). Expression of miRNAs was analyzed by real time
PCR as described previously.9 (link)
7
Verification of miRNA Expression in sEVs
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The miRNAs from the NGS panel were measured in the LI group Pre and Post-24h (n = 6) via qPCR to verify specificity of HI. Following miRNA isolation and purification from sEVs, cDNA was synthesized from miRNA utilizing a qScript miRNA cDNA Synthesis Kit (Cat no. 89168-790; Quantabio, Beverly, MA, USA). Next, cDNA was further enriched using PerfeCTa PreAmp SuperMix (Cat no. 89409-170; Quantabio) to increase cDNA template volume for qPCR application. qPCR assays were then performed using primer sets designed to specifically amplify candidate miRNAs identified through NGS as the most changed in the HI group: miR-92b-5p, miR-423-5p, miR-24-3p, miR-7844-5p, miR-144-5p, miR-221-5p, and miR-22-3p.
8
miRNA Extraction and cDNA Synthesis
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DNA LoBind products (Eppendorf) were used where possible. RNA extractions were performed with the Qiagen miRNeasy Mini Kit (Qiagen) using 50 μL sample volumes as previously described (13 (link), 14 ), the optional full speed centrifugation to remove residual buffer, and pre-heated ultrapure water (60°C) for two 30 μL elutions (60 μL total). First strand cDNA was prepared using the qScript miRNA cDNA Synthesis kit (Quantabio, Beverly, MA), using 7 μL RNA and the 20 min option for Poly(A) tailing as indicated in the vendor’s instructions. After reverse transcription (RT), samples were held at 4°C until ddPCR.
Frozen dose aliquots and plasma samples were thawed for miRNA extraction in animal-matched batches. Aliquots and samples in 1.5 mL microcentrifuge tubes were removed from storage at -80°C, and Qiazol reagent was added immediately. Samples were vortexed after thawing, incubated at RT for 5 min, then proceeded to RNA extraction.
Frozen dose aliquots and plasma samples were thawed for miRNA extraction in animal-matched batches. Aliquots and samples in 1.5 mL microcentrifuge tubes were removed from storage at -80°C, and Qiazol reagent was added immediately. Samples were vortexed after thawing, incubated at RT for 5 min, then proceeded to RNA extraction.
9
Quantitative RT-PCR for miRNA and mRNA Expression
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qRT-PCR was performed using the qScript miRNA cDNA Synthesis Kit (Quanta bio, Cat No. 95107-025) according to manufacturer’s instructions. Briefly, 2 μl of 500 ng/μl cDNA sample (diluted 1:10) was added to PCR tubes containing 18 μl of master mix (10 µl PerfeCTa SYBR Green SuperMix, 0.4 µl Custom miRNA Assay Primer (2 µM), 0.4 µl PerfeCTa Universal PCR Primer (10 µM), 7.2 µl Nuclease-Free Water). Samples were then run through the Rotor-Gene 3000 (Corbett Research) thermal cycler using custom designed primers (Tables 1 , 2 ). Cycling conditions involved initial holding step (95°C for 13 min), followed by 45 cycles of a two-step PCR (95°C for 15 s and 60°C for 60 s) and a dissociation phase. Fold-changes in miRNA and mRNA expression in treated samples compared to control samples were calculated using the 2−ΔΔCt method. RNU6 was used as a reference miRNA and the geometric mean of GAPDH and 18S rRNA (RNA18SN1) as reference RNA for HTR-8/SVneo and placental tissue samples.
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