Fasting blood samples were obtained from all subjects for extraction of mRNA. Total RNA was extracted from peripheral whole blood using the QIAamp RNA blood Mini Kit (QIAGEN, Chatsworth, CA, USA) following the manufacturer’s standard protocol. To ensure no DNA contamination, clean-up of the RNA was performed using QIAGEN spin columns with an additional DNAase step (QIAGEN, Chatsworth, CA, USA). Reverse transcription was performed using TaqMan reverse transcription reagents with random primers (Takara Bio, Kyoto, Japan). Samples were stored at -80 ºC prior to further use. 6 OCD patients and 4 healthy controls did not have mRNA successfully extracted. In all, 50 first episode and drug-free patients and 60 matched healthy controls were included.
Taqman reverse transcription reagents
Manufactured by Takara Bio
Sourced in Japan
The TaqMan Reverse Transcription Reagents are a set of reagents used for the reverse transcription of RNA to cDNA. These reagents include a reverse transcriptase enzyme, a random hexamer primer, and other necessary components for the reverse transcription reaction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Spin column, by Qiagen (1 mentions)
Qiaamp rna blood mini kit, by Qiagen (1 mentions)
Rneasy kit, by Takara Bio (1 mentions)
Viia7, by Thermo Fisher Scientific (1 mentions)
Power sybr green, by Takara Bio (1 mentions)
Primer premier 5, by Premier Biosoft (1 mentions)
Sybr green pcr mix, by Takara Bio (1 mentions)
Nano 300 microspectrophotometer, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Hieff qpcr sybr green master mix, by Yeasen (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Pet01, by MoBiTec (1 mentions)
6 protocols using taqman reverse transcription reagents
1
Blood mRNA Extraction and Purification
2
Quantitative Gene Expression Analysis
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Total RNA was extracted from the LA and the LSG using TRIzol reagent, according to the manufacturer’s protocol. Then, this was treated with DNase I to degrade any trace of DNA, and purified using an RNeasy Kit (Takara, Tokyo, Japan). The integrity of each sample was confirmed by agarose gel analysis. Total RNA was reverse transcribed using TaqMan reverse transcription reagents (Takara). The expression levels of candidate genes were measured by real-time quantitative RT-PCR (qRT-PCR) using Power SYBR Green (Takara) dye, and quantified using ViiA7 (Applied Biosystems, CA, USA). In each assay, both an endogenous control gene (β-actin) and a target gene from the same samples were amplified in duplicate in separate tubes. The mRNA levels of each target gene were calculated using the relative standard curve method and normalized against the corresponding β-actin mRNA levels. The expected amplicon sizes were confirmed by gel electrophoresis. The sequences of the genes studied were obtained from GenBank, and the primers were designed using Primer Premier 5.0 software (Premier Biosoft). Table 1 presents the primer sequences and amplicon sizes of the selected genes.
3
Profiling Cancer Invasion Genes
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Total mRNA from the lung adenocarcinoma cell lines was extracted using TRIzol reagent (Invitrogen) according to the manufacturer'sprotocol. 1 μg of mRNA was reverse transcribed to cDNA using Taqman reverse-transcription reagents (TAKARA, Japan). The primer sequences for the cancer invasion-related genes, including VEGFA, MMP2, TERT, PKLR, IGF1, RHOC, TGFB1, uPA, Id1, NFκB-p65, ERK1/2, IL1RL1, TWIST1, and ADAMTS1, are listed in Supplementary Table 1 . The PCR procedure for amplification was 95°C for 30 s, followed by 32 cycles of 94°C for 30 s, 50°C (uPA, IGF1, IL1RL1,PKLR, MMP2, TERT) or 55°C (VEGFA, NFκB-p65, TGFβ-1, ERK1/2, Id1, TWIST, ADAMTS1, RHOC, KLF17) for 30 s, and 72°C for 30 s. β-actin was used as a loading control.
4
Quantification of Autophagy Proteins in Kölliker's Organ
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About 4 μg of RNA were extracted from the Kölliker’s organ of 12 animals using the TRIzol reagents (ThermoFisher Scientific, Boston, MA, USA; Invitrogen #15596026,) according to the manufacturer’s instructions. The cDNAs were obtained using the TaqMan Reverse Transcription Reagents (Takara Bio Inc., Kusatsu, Japan). The reaction was carried out in the Applied Biosystem (Foster City, CA, USA), using the SYBR green PCR mix (Takara Bio Inc.). Each DNA sample was evaluated in triplicates. LC3-II, P62, Beclin1, Bcl2 and Caspase3 were the target genes to assess and β-actin was used as the endogenous reference. The primers were listed on Table 1 . The coefficient of variation (CV) values of the target genes and endogenous reference were calculated. The expression levels of mRNAs were calculated by the 2-DDCT method.
5
RNA Extraction and qPCR Analysis Protocol
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Cells were lysed with TRIzol (Invitrogen, #15596-026) for total RNA extraction. The concentration and purity of RNA were tested by a Nano300 microspectrophotometer (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesised using TaqMan Reverse Transcription Reagents (TaKaRa, #RR047A) according to the manufacturer’s instructions. Hieff qPCR SYBR Green Master Mix (Yeasen, #11201ES08) and gene-specific primers were used to perform qPCR as previously described.53 (link) The primers used in this experiment are presented in Supplementary Table 1 .
6
In Vitro Minigene Analysis of Genetic Variants
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The in vitro minigene analysis was performed as previously described (Booth et al., 2018 (link)). Wild-type and mutant minigene inserts were directly synthesized (Sangon Biotech, Shanghai, China, Supplementary Table S2 ) or amplified from the patients’ genomic DNA (Supplementary Table S3 ). The inserts were cloned into the pre-constructed exon-trap vectors pET01 (MoBiTec, Goettingen, Germany), pEGFP-C1 or pcMINI (Wuhan bioegle Biological Technology and Science Co., Ltd., Wuhan, China), all with intrinsic 3′ and 5′ exons separated by a multiple cloning site (MCS). The minigene constructs were then transfected into COS7 cells (ATCC_CRL1651)using LipofectamineTM3000 Transfection Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, United States). Cells were harvested 36 h after transfection. The total RNA was extracted using the Trizol method. cDNA was reversely transcribed by TaqMan Reverse Transcription Reagents (Takara Bio Inc., Japan). The spliced products were PCR amplified with intrinsic primers from the pET01, pEGFP-C1 or pcMINI vectors, detected by agarose gel electrophoresis and Sanger sequencing.
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