The total RNA was extracted from cells or tissues with TRIzol reagents (16096020 or AM1561; Thermo Fisher Scientific). A total of 5 μg RNA was reversely transcribed into cDNA according to the instructions of the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) kit (RR047A; Takara Bio Inc., Otsu, Shiga, Japan). RT-qPCR was then performed using the Fast SYBR Green PCR Kit (Applied Biosystems, Carlsbad, CA, USA) and the ABI PRISM 7300 RT-qPCR system (Applied Biosystems). All investigations involved at least 3 wells. The primers are shown in online suppl. Table 1 (for all online suppl. material, see www.karger.com/doi/10.1159/000518277 ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered as an internal reference, and the fold changes were calculated by relative quantification (2−ΔΔCt method).
Abi prism 7300 rt qpcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI PRISM 7300 RT-qPCR system is a real-time quantitative PCR (RT-qPCR) instrument. It is designed for gene expression analysis and quantification of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Fast sybr green pcr kit, by Thermo Fisher Scientific (6 mentions)
Primescript rt reagent kit, by Takara Bio (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rt qpcr kit, by Takara Bio (1 mentions)
Reverse transcription kit, by Promega (1 mentions)
B532451, by Sangon (1 mentions)
Fast sybr green mix, by Thermo Fisher Scientific (1 mentions)
Microrna reverse transcription kit, by EZBioscience (1 mentions)
Molpure cell tissue mirna kit, by Yeasen (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using abi prism 7300 rt qpcr system
1
RT-qPCR Protocol for Gene Expression
2
Quantitative Real-Time PCR of DLX6-AS1 Expression
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Total RNA was extracted using Trizol (15596026, Invitrogen Inc, Carlsbad, CA, USA). RNA was reversely transcribed to cDNA according to instructions in the PrimeScript RT reagent kit (RR047A, Takara, Tokyo, Japan). The synthesized cDNA was subjected to RT‐qPCR using the Fast SYBR Green PCR kit (Applied Biosystems, Oyster Bay, NY, USA) and the ABI PRISM 7300 RT‐qPCR system (Applied Biosystems, Oyster Bay, NY, USA). Triplicates were set for each well. With glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as the internal control, the fold changes of DLX6‐AS1 expression were calculated by means of the relative quantification (2−ΔΔCt method).10 Primer sequences are shown in Table 1 .
3
Extracellular Vesicle miRNA Profiling
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Abiding to the manufacturer’s protocols, miR Vana™ PARISTM RNA kits (AM1556, Invitrogen) was adopted for isolation of miRNA from extracellular vesicles (EVs), tissues, and cells. For qPCR of mRNA, cDNA was synthesized with the help of reverse transcription kits (M1701, Promega, Madison, WI). Afterwards, the obtained cDNA samples were warm bathed at 80 °C for 5 min to inactivate reverse transcriptase for subsequent PCR reaction. For miRNA qPCR, the primers in the kit were replaced. In the current experiment, miRNA first strand cDNA synthesis (tailing reaction) (b532451, Sangon, Shanghai, China) was adopted for reverse transcription. Finally, quantitative analysis of RNA was performed using Fast SYBR Green PCR kits (Applied Biosystems, NY) and an ABI Prism 7300 RT-qPCR system (Applied Biosystems), with three parallel wells set for each sample. U6 was employed as internal reference of miR-497 and GAPDH for other genes. 2−ΔΔCT method was finally adopted to measure the target gene relative expression. All primers (Additional file 1 : Table S1) were purchased from Sangon.
4
Quantitative RT-qPCR Analysis of ANGPTL4
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Total RNA from tissues or cultured cells was extracted using the Trizol (15596026, Invitrogen) extraction method and then reverse transcribed into cDNA using a PrimeScript RT reagent Kit (RR047A, Takara) according to the manufacturer's instructions. The Fast SYBR Green Mix (Applied biosystems) and its corresponding reaction system were applied to determine the level of target gene expression using an ABI PRISM 7300 RT-qPCR system (Applied biosystems), with previously reported reaction conditions 14 (link), 15 (link). The relative expression of ANGPTL4 to reference gene GAPDH was determined using the 2-ΔΔC method. The primers sequences are shown in Table S1 .
5
Profiling miRNA and mRNA Expression
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Total RNA from NSCs and hippocampal tissue samples was extracted employing Trizol reagent (Invitrogen, Carlsbad, CA), and miRNAs from cells and hippocampal tissue samples were extracted utilizing miRNA extraction kit (19331ES08, MolPure ® Cell/Tissue miRNA Kit, Yeasen, Shanghai, China). The extracted RNA was reversely transcribed into complementary DNA (cDNA) from 500 ng of RNA by referring to the PrimeScript RT reagent Kit (RR047A, Takara, Japan). miRNA was reversely transcribed into cDNA utilizing microRNA Reverse Transcription Kit (EZBioscience, EZB-Exo-RN1). The synthesized cDNA was subjected to RT-qPCR with the Fast SYBR Green PCR kit (4364344, Applied biosystems, Foster City, CA) and the ABI PRISM7300 RT-qPCR system (Applied biosystems). GAPDH served as a normalizer for mRNA, and U6 for miRNA. The relative expression of genes or mRNAs was analyzed employing the 2-ΔΔCt method. Primers are described in Table S1 .
6
Quantitative RT-PCR Protocol for RNA Analysis
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Total RNA was extracted using TRIzol reagents (15596026; Invitrogen) and reversely transcribed into complementary DNA (cDNA) according to the instructions of PrimeScript RT reagent Kit (RR047A; Takara Bio Inc). RT‐qPCR was performed on the synthesized cDNA using the Fast SYBR Green PCR kit (Applied Biosystems Inc) and an ABI PRISM 7300 RT‐qPCR system (Applied Biosystems). The relative expression of mRNA or miRNA was normalized to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) or U6, respectively, and was calculated using the 2−ΔΔCt method. The primer design is shown in Table 2 .
7
Gene Expression Analysis Using RT-qPCR
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Total RNA content was extracted from the cells using the TRIzol reagent (15596026, Invitrogen Inc., Carlsbad, CA, USA), and then reverse-transcribed into complementary DNA (cDNA) according to the instructions of the PrimeScript RT reagent kit (RR047A, Takara, Japan) at 37°C for 30–50 min and 85°C for 5 s. Subsequently, RT-qPCR was performed using Fast SYBR Green PCR kits (Applied Biosystems Inc. Carlsbad, CA, USA) on the ABI PRISM 7300 RT-qPCR system (Applied Biosystems). Triplicate wells were set for all investigations. The primer sequences are shown in Supplementary Table 2 . The fold changes were calculated by means of relative quantification (2−ΔΔCt method) with GADPH serving as the loading control.
8
Quantification of Plasma miRNA Expression
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The reverse transcription reaction was conducted using a TaqMan microRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) in a 15 µl solution consisting of 5 µl RNA template, 1.5 µl 10X reverse transcription buffer, 1 µl MultiScribe reverse transcriptase, 0.15 µl of 100 mM dNTPs, 0.19 µl of RNase inhibitor (20 U/µl), 1 µl of gene-specific primer, and supplemented with nucleasefree water. For the synthesis of complementary DNA (cDNA), the reaction mixtures were incubated at 16˚C for 30 min, at 42˚C for 30 min, and at 85˚C for 15 min and then held at 4˚C.
RT-qPCR was used to quantify the expression levels of the miRNAs. Each amplification reaction was carried out in a final volume of 20 µl containing 2 µl of the cDNA (100 ng), 10 µl of TaqMan 2X Universal PCR Master Mix with No AmpErase UNG (Applied Biosystems), 1 µl of gene-specific primer, and 7 µl of nuclease-free water. The ABI Prism 7300 RT-qPCR system (Applied Biosystems) was used to detect the plasma miRNA levels. The PCR conditions consisted of an initial polymerase activation step at 95˚C for 10 min, 46 cycles of 95˚C for 15 sec and 60˚C for 1 min. Each reaction was performed in triplicate. The expression levels of the miRNAs in plasma were normalized to the levels of small nuclear RNU6B (U6) and were calculated using the 2 -ΔΔCt method, as previously described (24) .
RT-qPCR was used to quantify the expression levels of the miRNAs. Each amplification reaction was carried out in a final volume of 20 µl containing 2 µl of the cDNA (100 ng), 10 µl of TaqMan 2X Universal PCR Master Mix with No AmpErase UNG (Applied Biosystems), 1 µl of gene-specific primer, and 7 µl of nuclease-free water. The ABI Prism 7300 RT-qPCR system (Applied Biosystems) was used to detect the plasma miRNA levels. The PCR conditions consisted of an initial polymerase activation step at 95˚C for 10 min, 46 cycles of 95˚C for 15 sec and 60˚C for 1 min. Each reaction was performed in triplicate. The expression levels of the miRNAs in plasma were normalized to the levels of small nuclear RNU6B (U6) and were calculated using the 2 -ΔΔCt method, as previously described (24) .
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