TRIzol reagent (Sigma-Aldrich) was used to extract total RNA from the A431 cells after stimulation by LL-37. The total RNA (3 µg) was reverse transcribed to cDNA in a total volume of 20 µl using a reverse transcription reaction kit (Promega Corporation, Madison, WI, USA). qPCR was performed using an Mx 3000P Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. SYBR Premix Ex Taq II (Takara Biotechnologies Co., Ltd., Dalian, China) was used as a DNA-specific fluorescent dye. PCR was performed for 50 cycles of 95°C for 10 sec and 60°C for 30 sec. Primer sequences for the detection of mRNA expression were synthesized as follows: DbpA specific primers, 5-CTCTACAGTTTCTCCATCTCCTAC-3 (forward) and 5-TTCTCGCCACCAAAGTCCT-3 (reverse); and human β-actin primers, 5-TTCCATATCGTCCCAGTTGGT-3 (forward) and 5-CCAGGGCGTTATGGTAGGCA-3 (reverse). The dbpA transcriptional level was corrected based on the corresponding level of β-actin transcription. All the values are from the results of at least three independent experiments.
Reverse transcription reaction kit
Manufactured by Promega
Sourced in United States
The Reverse Transcription Reaction Kit is a laboratory tool designed for the conversion of RNA to complementary DNA (cDNA). It contains the necessary components, including reverse transcriptase enzyme, buffers, and primers, to facilitate this critical step in gene expression analysis and other RNA-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Merck Group (2 mentions)
Mx 3000p real time pcr system, by Thermo Fisher Scientific (1 mentions)
Qsybr green containing pcr kit, by GenePharma (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Mx3000p qpcr system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Express sybr greener qpcr supermix universal kit, by Thermo Fisher Scientific (1 mentions)
Rotor gene 6000 system, by Qiagen (1 mentions)
4 protocols using reverse transcription reaction kit
1
Quantifying DbpA mRNA Expression
2
Quantification of miR-124 and PRRX1 mRNA
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Total RNA was extracted from cells using TRIzol reagent (Invitrogen, USA). For miR-124, reverse transcription and qRT-PCR reactions were performed using a qSYBR-green-containing PCR kit (GenePharma, Shanghai, China). U6 snRNA was used as an endogenous control. The precursor form of miR-124 was amplified. For detecting PRRX1 mRNA, cDNA was synthesized from 1 μg of total RNA using the reverse transcription reaction kit according to the manufacturer's instructions (Promega, USA). Human GAPDH was amplified in parallel as an internal control. The primers were listed in Supplementary Table S1 . All these samples were normalized to internal controls and fold changes were calculated through relative quantification (2-ΔΔCt).
3
RNA Extraction and qPCR Analysis of YB-1 Expression
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TRIzol reagent (Sigma-Aldrich; Merck KGaA) was used to extract total RNA from cells. Total RNA (3 µg) was then reverse transcribed to cDNA in a total volume of 20 µl using a reverse transcription reaction kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. Then, qPCR was performed using an Mx 3000P qPCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. SYBR Premix Ex Taq II (Takara Biotechnology Co., Ltd., Dalian, China) was used as a DNA-specific fluorescent dye. PCR was performed for 50 cycles of 95°C for 10 sec and 60°C for 30 sec. Primer sequences for detection of mRNA expression were synthesized as follows: YB-1 specific primers, 5-CAGAATAGTGAGAGTGGGG-3 (forward) and 5-ATGTAGTAAGGTGGGAACC-3 (reverse); human β-actin primers, 5-TTCCATATCGTCCCAGTTGGT-3 (forward) and 5-CCAGGGCGTTATGGTAGGCA-3 (reverse). The YB-1 transcriptional level was normalized against the transcriptional level of β-actin. All values are from the results of at least three independent experiments. Relative mRNA expression levels were calculated using the 2−∆∆Cq method (25 (link)).
4
Quantification of TIM-1 and TIM-3 Expression
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One microgram of total RNA was reverse transcribed to cDNA in a 20 μL total volume system using a reverse transcription reaction kit (Promega). Real-time PCR was performed using the Express SYBR GreenER qPCR Supermix Universal Kit (Invitrogen) on a Rotor-gene 6000 system (Qiagen). The 25-μL PCR mixture contained 2 μL reverse-transcribed product, 12.5 μL SYBR GreenER Supermix, 8.5 μL RNase-free water, 1 μL forward, and 1 μL reverse primers. The reaction was performed on a 72-well optical plate on triplicate. The first step of PCR protocol was 95°C for 10 s, followed by 40 cycles of 95°C for 5 s, and the second step was 60°C for 30 s. A melting-curve analysis was performed to ensure specificity of the PCR products, all of which were subjected to electrophoresis on an agarose gel for confinement to a single band of the expected size. The expression of TIM-1 and TIM-3 was normalized to β-actin and determined using the comparative (2 -ΔΔCt ) method (Livak and Schmittgen, 2001) . Primers were as follows: TIM-1 forward: 5'-GCTTTGCAAAATGCAGT TGA-3', and reverse: 5'-TGTT-GAATGCCAGATGAAA-3'; TIM-3 forward: 5'-GACTTCAC TGCAGCCTTTCC-3', and reverse: 5'-GATCCCTGCTCCGATGTAGA-3'; β-actin forward: 5'-TACAGCTTCACCACCACAGC-3', and reverse: 5'-AAGGAAGGCTGGAAAAGAGC-3'.
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