Reverse transcription reactions were carried out using miRNA First-Strand cDNA Synthesis SuperMix (TransScript) according to the manufacturer’s instructions. A SYBR PrimeScript miRNA RT-PCR Kit was used to perform qRT-PCR in a fluorescence detection system (TianGen Biotech, Beijing, China) following the manufacturer’s instructions. All reactions were performed in triplicate for each sample. The 2-∆∆CT method was then used to calculate the relative expression levels of the miRNAs [74 (link)]. Target genes of the miRNAs that were differentially expressed during grain development in the HN and LN treatments were selected to validate their expression patterns in developing wheat grain via qRT-PCR. Fisher’s least significant difference test (LSD) was subsequently used to distinguish differences in relative expression levels between different development stages using SPSS (Statistical Program for Social Science) software; P < 0.05 was considered statistically significant. All primer sequences used and the target gene identification results are given in Table S7 (Additional file 8 ).
Sybr primescript mirna rt pcr kit
Manufactured by Tiangen Biotech
Sourced in China
The SYBR PrimeScript miRNA RT-PCR Kit is a laboratory equipment product designed for the detection and quantification of microRNA (miRNA) expression using reverse transcription and real-time PCR technology. The kit includes reagents for reverse transcription and real-time PCR amplification, allowing for the sensitive and accurate measurement of miRNA levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence detection system, by Tiangen Biotech (2 mentions)
Mircute mirna first strand cdna synthesis kit, by Tiangen Biotech (2 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase 1 treatment, by Promega (1 mentions)
Sybr green real time pcr master mix, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
M mlv rtase cdna synthesis kit, by Takara Bio (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
Rnase free dnase 1, by Promega (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
5 protocols using sybr primescript mirna rt pcr kit
1
Evaluating miRNA Expression Patterns in Wheat Development
2
miRNA and Gene Expression Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs were extracted from the fourth leaves of the WT and phyB plants as described above. Genomic DNA contamination was eliminated by RNase-free DNase I treatment (Promega, Madison, WI, USA). For miRNA qRT-PCR, miRNA first-strand cDNA was synthesized using the miRcute miRNA First-Strand cDNA Synthesis kit (Tiangen, Beijing, China). The expression levels of 15 random selected miRNAs were quantified by qRT-PCR with the SYBR PrimeScript™ miRNA RT-PCR kit (Tiangen, Beijing, China). For targets qRT-PCR, first-strand cDNA was synthesized using M-MLV reverse transcriptase according to the manufacturer's instructions (Promega, Madison, WI, USA). The expression patterns of selected genes were detected using SYBR Green Real-time PCR Master Mix (PE Applied Biosystems, Foster City, CA, USA) on the Stepone Plus system (Applied Biosystems, USA). The primers used in qRT-PCR are listed in Additional file 1 . Three biological replicates were included for each miRNA and target gene in qRT-PCR analysis. The relative expression ratios of each miRNA and target gene were calculated using the delta-delta threshold cycle relative quantification method with the internal control of 5.8s rRNA and OsEF-1α, respectively.
3
Quantifying miRNA and Precursor Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA (including miRNA) was extracted using the miRNeasy Mini kit (Qiagen). Residual genomic DNA was eliminated with RNase‐free DNase I (Promega). To analyze miR530 expression, miRNA first‐strand cDNA was synthesized with the miRcute miRNA First‐Strand cDNA Synthesis kit (Tiangen, Beijing, China). The expression of miR530 was then quantified by qRT‐PCR with the SYBR PrimeScript™ miRNA RT‐PCR kit (Tiangen) with 5.8S rRNA as the internal control. To analyze the expression of the OsmiR530 precursor, the target mimic and OsPL3, c. 2 μg total RNA for each sample was reverse‐transcribed with the M‐MLV RTase cDNA synthesis kit (TaKaRa, Dalian, China). The resulting cDNA was used as the template for a qRT‐PCR assay, which was completed with the SYBR Green PCR master mix (TaKaRa). The OsEF‐1α gene was used as the internal control. The qRT‐PCR analysis involved three biological replicates for each miRNA and gene. The relative expression ratios of OsmiR530, its precursor OsMIR530 and OsPL3 were calculated according to the delta‐delta threshold cycle relative quantification method. Details regarding the qRT‐PCR primers are provided in Table S1 .
4
qPCR Quantification of miRNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The qPCR reactions were performed with a SYBR PrimeScript miRNA RT-PCR Kit on a Fluorescence detection system (TianGen Biotech, Beijing, China). Each 20 μl reaction contained 1 μl cDNA template (∼100 ng), 1 μl 10 μM PCR forward primer, 1 μl 10 μM Uni-miR qPCR primer, 10 μl 2× SYBR premix EX TaqII, and 7 μl ddH2O. The amplification reactions were performed by first incubating at 95°C for 5 min, followed by 45 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 45 s. Following amplification, a threshold was set and the threshold cycle (CT) was recorded automatically. All reactions were performed in triplicate for each sample. The relative expression levels of the miRNAs were calculated using the 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)) and the data were normalized to the CT values for the Actin gene. The primer sequences for 16 differentially expressed miRNAs are given in Supplementary Table S13-1 .
5
Quantifying miRNA Expression in Drosophila Gonads
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The experiments were performed on a CFX connect™ real-time system (BioRad) with three biological replicates and two technical replicates. Forty pairs of 4-day-old virgin female ovaries and sixty pairs of 1-day-old male testes were dissected for each biological replicate. Total RNA was extracted with a miRNeasy extraction kit (TIANGEN catalog no. DP501) together with an RNase-Free DNase set. RNA quality was estimated with a Nanodrop spectrophotometer and by agarose gel electrophoresis. The expression of miRNA was checked by using the SYBR Prime-Script miRNA RT-PCR Kit (TIANGEN catalog no. FP401). Total RNA (2 μg) was reverse transcribed, and 30 ng cDNA products were added to a 20-μL quantification system. The primers used in this study are shown in Additional file 6 . The reactions were incubated at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 65 °C for 34 s. A dissociation curve was obtained to ensure that only one product was amplified after the amplification phase. The relative expression of each miRNA was normalized against the reference gene (U6 snRNA) using 2-ΔCT (ΔCT = CT, target miRNA - CT, U6 snRNA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!