Stem loop real time pcr mirna kit

Total RNA was extracted from exosomes by SeraMir (System Biosciences) following the manufacturer's instructions and cellular RNA was extracted using the RNAprep pure cell/bacteria kit (TIANGEN, Beijing, China). The purity of the isolated RNA was determined at OD260/280 using a Nanodrop ND-1000 (Thermo Scientific), and integrity was assessed by 1% agarose gel electrophoresis. RNAs were reverse-transcribed to first-strand cDNA using the PrimeScipt RT Reagent Kit (TaKaRa, Dalian, China). The cDNA was subjected to qRT-PCR using the SYBR kit (TaKaRa, Dalian, China). miR-128-3p expression was quantified with a stem-loop real-time PCR miRNA kit (Ribobio, Guangzhou, China). The threshold cycle (Ct) was determined for each reaction using the 2^−ΔΔCt method, and the expression of each gene of interest was normalized to that of the endogenous control gene (U6 for miRNAs or GAPDH for mRNAs). The primer sequences in the study are shown in Supplementary Table 1.

The TRIzol® Reagent (Invitrogen, CA, USA) was used for extraction of cultured cell lines’ total RNA which was then reverse transcribed into cDNA with a specific stem-loop real-time PCR miRNA kit (RiboBio, Guangzhou, China). The SYBR Green qPCR system (Takara, Dalian, CHN) was utilized in the a/1uantitative real-time PCR (qPCR) procedure with GAPDH acting as an internal control. qPCR was carried out with the Applied Biosystems 7900HT real-time PCR system.

CFs were treated and collected as described above. Total RNA was extracted from CFs and heart tissues by Trizol reagent (TaKaRa, Tokyo, Japan). First strand complementary DNA (cDNA) was synthesized using the Reverse Transcription Kit (TaKaRa, Tokyo, Japan) according to manufacturer’s instructions. For quantitative PCR (qPCR), PCR amplifications were quantified using the SYBR Green PCR Master Mix (Applied Biosystems) and normalized to GAPDH gene expression. MiR-33a quantification was performed with a stem-loop real-time PCR miRNA kit (Ribobio, Guangzhou, China).
The sequences of the q-PCR primers are described below: miR-33a (forward: 5′-GATCCTCAGTGCATTGTAGTTGC-3′; reverse: 5′-CTCTGTCTCT CGTCTTGTTGGTAT-3′), Col1A1 (forward: 5′-TCCTGACGCATGGCCAAGAA-3′; reverse: 5′-CATAGCACGCCATCGCACAC-3′), Col3A1 (forward: 5′-TGGACAGATGCTGGTGCTGAG -3′; reverse: 5′-GAAGGCCAGCTGTACATCAAGGA-3′), CTGF (forward: 5′-GCAGCTAGAGAAGCAGAGC-3′; reverse: 5′-GGTGCAGCCAGAAAGCTC-3′), MMP16 (forward: 5′-AATCTCCTCAGGGAGCATTTGTA-3′; reverse: 5′-TCCAGGTTCTACC TTGAGTATCTG-3′), U6 (forward: 5′-ATTGGAACGATACAGAGAAGATT-3′; reverse: 5′-GGAACGCTTCACGAATTTG-3′), and GAPDH (forward: 5′-GAC ATG CCG CCT GGA GAA AC-3′; reverse: 5′-AGC CCA GGA TGC CCT TTA GT-3′).

Total RNA from six samples (three goats) was extracted with TRIzol (Invitrogen, CA) according to the manufacturer’s instructions. Total RNA was reverse-transcribed to cDNA using the PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan) and RT-qPCR was performed using SYBR Premix Ex Taq (TaKaRa, Tokyo, Japan). The mRNA primers were synthesized by Shanghai Shenggong, China (Supplementary Table 9). The relative mRNA levels were normalized to β-actin (Hou et al., 2016 (link)) expression for each sample; For miRNA detection, reverse transcription followed by stem-loop RT-qPCR was performed according to the manufacturer’s protocols using the Bulge-LoopTM miRNA RT-qPCR Primer (RiboBio, Guangzhou, China). Quantification of miRNA was performed with a stem-loop real-time PCR miRNA kit (RiboBio). The relative miRNA levels were normalized to U6 small nuclear RNA (Carvalho et al., 2019 (link)) expression for each sample.

RNA extraction and real‐time quantitative real‐time polymerase chain reaction (PCR) analysis were performed as mentioned earlier.⁴² For microRNA quantification, total RNA was reverse‐transcribed by TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems), and then, TaqMan miRNA assay was performed according to the manufacturer's agreement (Applied Biosystems). Quantification of miR‐21‐5p was performed with a stem‐loop real‐time PCR miRNA kit (Ribobio). U6 was used as the internal reference for qRT‐PCR.

Total RNA was extracted from freshly isolated lysates of mouse testis and brain using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s recommendations. For miRNA analysis, total RNA was reverse transcribed into cDNA using a specific stem-loop real-time PCR miRNA kit (Ribobio, Guangzhou, China). RT- PCR was performed using the Platinum SYBR Green qPCR SuperMix-UDG system (Invitrogen, CA, USA) on an Applied Biosystems 7900 system. The miR-124 primers are shown below. PCR products were separated by electrophoresis on 2% agarose gels.