Total RNA content was extracted from the human and mouse NP tissues and cells by means of TRIzol kits (Invitrogen). The obtained total RNA (including miRNAs and mRNAs) was then reverse-transcribed into complementary DNA (cDNA) using a TaqMan™ MicroRNA Reverse Transcription Kit (4366596, Thermo Fisher Scientific, Waltham, MA, USA) and High-Capacity cDNA Reverse Transcription Kit (4368813, Thermo Fisher Scientific), respectively. Subsequently, RT-qPCR was carried out with the SYBR® Premix Ex Taq™ (Tli RNaseH Plus) reagent kit (RR820A, Takara Bio Inc., Otsu, Shiga, Japan) on an ABI7500 real-time quantitative PCR system (ABI Company, Oyster Bay, NY, USA). The employed primer sequences are presented in Supplementary Table 2 . U6 was employed as the internal control for miRNA quantification, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) utilized for mRNA quantification. The fold changes were calculated by means of relative quantification (the 2-ΔΔCt method) [27 (link)].
Sybr premix ex taq tli rnaseh plus reagent kit
Manufactured by Takara Bio
Sourced in Japan
SYBR® Premix Ex Taq™ (Tli RNaseH Plus) is a ready-to-use reagent kit for real-time PCR amplification. It contains a DNA polymerase, SYBR® Green I dye, and other necessary components for efficient and sensitive detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Trizol kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Gdna eraser, by Takara Bio (1 mentions)
Abi 7500 real time quantitative pcr system, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
2 protocols using sybr premix ex taq tli rnaseh plus reagent kit
1
Quantifying RNA Expression in NP Tissues
2
Quantitative Real-Time PCR Analysis of miRNA and mRNA Expression
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Total RNA in cells was extracted using a TRIzol kit (Invitrogen, Carlsbad, CA, USA). RNA concentration was determined by NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). RNA (1 μg) was then reverse transcribed into the cDNA following the instructions of PrimeScript RT Reagent Kit by a gDNA Eraser (Takara Bio, Tokyo, Japan). qRT-PCR was performed using a SYBR Premix Ex Taq (Tli RNaseH Plus) reagent kit (Takara Bio, Tokyo, Japan) by an ABI7500 real-time quantitative PCR system (Thermo Scientific, Wilmington, DE, USA). Primer sequences (Shanghai GenePharma, Shanghai, China) are listed in Table 1 . β-actin or U6 was used as endogenous controls. Fold changes in expression were calculated by the 2−ΔΔCt method.34 (link)
Primer Sequences for qRT-PCR
| Gene | Sequence |
|---|---|
| miR-9-3p | F: 5′-GGAGACCGGAAATGTAGCCA-3′ |
| R: 5′-AATGGCCCGTGGAGTCTTTG-3′ | |
| U6 | F: 5′-CTCGCTTCGGCAGCACA-3′ |
| R: 5′-AACGCTTCACGAATTTGCGT-3′ | |
| ESM1 | F: 5′-TCAGCGAGTACTTCCTAAAT-3′ |
| R: 5′-TCTCCTTC TAGAGCGTTACA-3′ | |
| GAPDH | F: 5′-TTCACCACCATGGAGAAGGC-3′ |
| R: 5′-GGCATGGACTGTGGTCATGA-3′ |
ESM1, endothelial cell-specific molecule 1; F, forward; GAPDH, glyceraldehyde phosphate dehydrogenase; miR-9-3p, microRNA-9-3p; R, reverse.
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