All in one mirna qpcr kit

Quantitative reverse transcription-polymerase chain reaction (PCR) was performed for miRNA quantification using All-in-One miRNA First-Strand cDNA Synthesis Kit and All-in-One miRNA qPCR Kit (GeneCopoeia, MD, USA). The probes were purchased from Invitrogen (Invitrogen, CA, USA). The sequences are listed in Table 1. All reactions were run in triplicate on the Stratagene Mx3005p Real-Time qPCR System (Agilent Technologies, CA, USA). The average levels of U6 small nuclear RNA and two miRNAs (miR-634 and miR-1228-3p), which were not differentially expressed in the microRNA microarray, were used as internal control in plasma miRNA analysis. The differential expression level of plasma miRNA was calculated by the following equation: $\begin{array}{c}\mathrm{\Delta }\mathrm{C}{\mathrm{t}}_{\mathrm{m}\mathrm{i}\mathrm{R}}=\mathrm{C}{\mathrm{t}}_{\mathrm{m}\mathrm{i}\mathrm{R}}-\frac{\left(\mathrm{C}{\mathrm{t}}_{\text{miR-U6}}+\mathrm{C}{\mathrm{t}}_{\text{miR-634}}+\mathrm{C}{\mathrm{t}}_{\text{miR-1228}}\right)}{\mathrm{3}}\\ \\ \mathrm{\Delta }\mathrm{\Delta }\mathrm{C}\mathrm{t}=\mathrm{\Delta }\mathrm{C}{\mathrm{t}}_{\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}}-\mathrm{\Delta }\mathrm{C}{\mathrm{t}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}.\\ \end{array}$ Fold changes were calculated through relative quantification: 2^−ΔΔCt [21 (link)].

Total RNAs from tissues and cultured cells were extracted using Trizo reagent (Invitrogen, USA) according to the manufacturer’s protocol. cDNA was reversely transcribed by using PrimeScriptTM RT Master Mix (Perfect Real Time) (TaKaRa Biotechnology, China). All-in-One™ miRNA First-Strand cDNA Synthesis Kit (Genecopoeia, China) was used for miRNA reverse transcription and RT-qPCR was performed using All-in-One™ miRNA qPCR Kit (Genecopoeia, Guangzhou, China) of miR-200a (Cat#HmiRQP0298), miR-200b (Cat#HmiRQP0300), miR-200c (Cat#HmiRQP0302), miR-141(Cat#HmiRQP0184), miR-429 (Cat#HmiRQP0497) and U6 (Cat#HmiRQP9001). Real-time PCR was performed with SYBR Green detection chemistry (TaKaRa Biotechnology, China) on ABI 7500 Real-Time PCR System (Applied Biosystems). For relative quantification, 2^-ΔΔCt was calculated, with U6 RNA as a reference in miRNA analysis and GAPDH as a reference in the analysis of protein coding genes. The indicated primers were placed in Additional file 2: Table S2.

Total RNA was isolated from cells with a RNA isolation Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. First-strand cDNA synthesis was synthesized with the first-strand synthesis system (Thermo Scientific). Real-time PCR was carried out using an ABI 7500 with SYBR Green detection (Applied Biosystems, Foster City, CA, USA) by the CFX96 Real-Time PCR System (Bio-Rad iQ5 program). GAPDH was used as an internal control. miRNAs were isolated from cultured cells and purified with the miRCURY RNA isolation Kit (Exiqon, Vedbaek, Denmark). cDNA was generated with the All-in-One™ miRNA First-Strand cDNA Synthesis Kit (GeneCopoeia, Guangzhou, China), and quantitative real-time PCR (qRT-PCR) was performed by using the All-in-One™ miRNA qPCR Kit (GeneCopoeia) according to the manufacturer’s instructions. The miRNA sequence-specific RT-PCR primers and the endogenous control RNU6 were purchased from GeneCopoeia. The relative quantitative expression was calculated by normalizing the results with RNU6. Primer sequences are provided in Supplementary Table 1.

Real-time PCR of mRNA and miRNA was performed using SYBR Green PCR Master Mix (Fermentas, Guangzhou, China) and All-in-One™ miRNA qPCR Kit (GeneCopoeia, Maryland, USA) respectively, according to the manufacturer’s instructions. The experiments were repeated at least in triplicates. The primers for Real-time PCR are shown in Additional file
8: Table S8.

Total RNAs were extracted from cells and from SHED-Exos using the Trizon reagent (Cat. 50,175,111, Thermo Fisher Scientific). RNA concentrations were measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Shanghai, China), and each RNA was then reverse transcribed to complementary DNA (cDNA) using a superscript III first strand kit (Cat. 18,080–05, Invitrogen). qRT-PCR reactions were performed with the SYBR Green qPCR Mix (Cat. 9211, Biosharp) in a total volume of 10 μl using a LightCyclerR 96 (Roche Diagnostics, Rotkreuz, Switzerland) according to the manufacturer’s instructions, with the following parameters: 95 °C for 30 s, 40 cycles at 9 °C for 5 s, at 60 °C for 20 s and ended with an elongation step for 15 s at 72 °C. GAPDH was used as an internal control. The PCR primers used are shown in the Supplementary Table.
Similarly, following cDNA synthesis using an All-in-one™ miRNA first-stand cDNA synthesis kit (Cat. AMRT-0020, GeneCopoeia), miRNA qRT-PCR reactions were performed using the All-in-one™ miRNA qPCR kit (Cat. AMRP-1200, GeneCopoeia) in a total volume of 10 μl. U6B small nuclear RNA (RNU6B) was chosen to normalize the expression of miRNAs [26 (link)]. The relative expression levels were determined using the 2^−ΔΔCt method. All primers used for miRNA qRT-PCR were purchased from GeneCopoeia, Guangzhou, China.

miRNA was extracted from HSCR stenotic colon segments and control colon tissue from 76 HSCR patients using SanPrep Column microRNA Mini-Preps Kit (Sangon Biotech, Shanghai, China). miRNAs were reverse transcribed into cDNA using the All-in-One™ miRNA First-Strand cDNA Synthesis Kit (GeneCopoeia Inc., Rockville, MD, USA). Realtime PCR was performed using the All-in-One™ miRNA qPCR Kit (GeneCopoeia). The primers were included in S1 Table and S2 Table.