In order to clarify the expression level of tiRNA-Gly-GCC-1 and TLR4, SYBR-based RT-qPCR was conducted in tissues and cell lines. In brief, total RNA was collected from 6-well plates (3 wells per group) and 6 pairs of tissues (tumor and paracancerous tissues) using Trizol. After RNAs’ concentration and purity and integrity were verified by NanoDrop ND-1000 and gel electrophoresis, RNA pre-treatments including 3’ terminal deacetylation and demethylation were performed for tiRNA-Gly-GCC-1 using the rtStar™ tRF&tiRNA Pretreatment Kit (Cat# AS-FS-005, Arraystar). Next, complementary DNA (cDNA) was synthesized using the rtStar™ First-Strand cDNA Synthesis Kit (3′ and 5′ adaptors) (Cat# AS-FS-003, Arraystar). qRT-PCR was performed in the QuantStudio™ 5 Real-time PCR System (Applied Biosystems) with a 2× PCR master mix (AS-MR-006-5). All indicators were carried out according to the following procedure: 95 °C denaturation (10 min), 95 °C (10 s), and 60 °C (60 s), followed by 40 cycles. After the amplification reaction, the melting curve procedure was performed. The 2−ΔΔCT method was used. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for the endogenous control gene. All reactions were performed in triplicate.
Rtstar first strand cdna synthesis kit 3 and 5 adaptor
Manufactured by Arraystar
Sourced in United States
The RtStar™ First-Strand cDNA Synthesis Kit is designed for the conversion of RNA to cDNA. It includes 3' and 5' adaptors for subsequent applications.
Automatically generated - may contain errors
Lab products found in correlation
Pcr master mix, by Arraystar (10 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (8 mentions)
Rtstar trf tirna pretreatment kit, by Arraystar (6 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (5 mentions)
Rtstar trf and tirna pretreatment kit, by Arraystar (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Agilent dna 1000 chip kit, by Agilent Technologies (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Prism v8, by GraphPad (1 mentions)
Trizol, by Tiangen Biotech (1 mentions)
Thunderbird next sybr qpcr mix, by Toyobo (1 mentions)
Revertra ace qpcr rt master mix, by Toyobo (1 mentions)
M mulv reverse transcriptase, by Qiagen (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
16 protocols using rtstar first strand cdna synthesis kit 3 and 5 adaptor
1
Quantitative Analysis of tiRNA-Gly-GCC-1 and TLR4 Expression
2
RNA Reverse Transcription Protocol
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Pre-processed RNAs underwent reverse transcription using the rtStar ™ First-Strand cDNA Synthesis Kit (3' and 5' adaptors) (Arraystar) according to manufacturer instructions.
3
Small RNA Library Preparation
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The pretreated total RNA was used to prepare the sequencing library by rtStar™ First-Strand cDNA Synthesis Kit (3’ and 5’ adaptors) (Cat#: AS-FS-003, Arraystar, MD, USA) in the following steps: 1) 3'-adapter ligation; 2) 5'-adapter ligation; 3) cDNA (complementary DNA) synthesis; 4) PCR amplification; 5) size selection of 135~160bp PCR amplified fragments (corresponding to 15~40nt small RNA size range). The prepared tRF&tiRNA-seq libraries were finally quantified using BioAnalyzer2100(Agilent, California, USA) by Agilent DNA 1000 chip kit (Agilent, part # 5067-1504) to quantified.
4
Validating tsRNA Expression Changes via qRT-PCR
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To verify treatment-induced changes in tsRNAs via RNA-seq, all tsRNAs were selected, and their expression levels were detected by qRT-PCR. First, total RNA was reverse transcribed into cDNA using the rtStar First-Strand cDNA Synthesis Kit (3′ and 5′ adaptors; Arraystar) according to the manufacturer’s protocol. Then, qRT-PCR amplification was performed using a Quant-Studio 5 Real-Time PCR System (Applied Biosystems) and 2 × PCR master mix (Arraystar). The amplification conditions were as follows: incubation at 95°C for 10 min and then 40 cycles of 95°C for 10 s, 60°C for 60 s, and 95°C for 15 s. The relative tsRNA expression level was calculated using the 2–ΔΔCt method and was normalized to U6 as a housekeeping gene. Specific primers for each gene are listed in Supplementary Table S1 . All reactions were performed in triplicate.
In addition, target mRNAs for the treatment of related tsRNAs were also verified by qRT-PCR. One target per tsRNA was randomly selected. GAPDH was used as an internal control to normalize the data.
In addition, target mRNAs for the treatment of related tsRNAs were also verified by qRT-PCR. One target per tsRNA was randomly selected. GAPDH was used as an internal control to normalize the data.
5
Small RNA Library Preparation
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To further improve the quality of the extracted RNA, the rtStar™ tRF&tiRNA Pretreatment Kit (Arraystar, Rockville, MD, USA) was used to remove small RNAs that would interfere with qPCR. The rtStar™ First-Strand cDNA Synthesis Kit (3′ and 5′ adaptor) (Cat# AS-FS-003, Arraystar, USA) was then used to create a cDNA library of small RNAs, which were subjected to 3′-terminal deacylation, 3′-cP removal, and 5′-P addition, demethylation, ligation of the 3′ adaptors, hybridization of reverse transcription primers, ligation of the 5′ adaptors, and finally reverse transcription into cDNA.
6
Quantitative Analysis of tRNA-Derived Small RNAs
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Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to confirm the selected tsRNAs' sequencing results. RNA was treated by rtStar™ tRF&tiRNA Pretreatment Kit (Arraystar, Rockville, MD, USA) and then converted to cDNA using rtStar™ First-Strand cDNA Synthesis Kit (3' and 5' adaptor) (Arraystar). qRT-PCR experiments were performed on ViiA 7 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) by using 2 × PCR Master Mix (Arraystar). Reaction conditions were described in our previous study 29 (link). The 2-ΔΔCt method 30 (link) was applied in the calculation of tsRNA expression levels normalized with U6. The primers of tested tsRNAs were listed in Table 1 .
7
qRT-PCR Validation of Differentially Expressed tRFs
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Differentially expressed tRFs were selected to perform qRT‐PCR validation. Total RNA was extracted from 12 pairs CCA and adjacent normal samples by TRIZOL (TIANGEN BIOTECH). Total RNA was pretreated by rtStar™ tRF&tiRNA Pretreatment Kit (Cat# AS‐FS‐005). cDNA was synthesized with the rtStar™ First‐Strand cDNA Synthesis Kit (3' and 5' adaptor) (Cat#AS‐FS‐003‐02; Arraystar). The primers were designed for Predesigned Human tRNA Primer Sets V2.0. U6 was utilized as an internal control. Quantitative Real‐time PCR was performed by Arraystar SYBR Green Real‐Time qPCR Master Mix (Cat#AS‐MR‐006‐5). The relative expression level of each tRNA‐derived fragments was calculated with 2−ΔCt. SPSSv24.0 (IBM; SPSS) and GraphPad Prism V8.0 (GraphPad software) were used to perform statistical analysis. p < 0.05 was statistically significant.
8
Validation of Treatment-Related tsRNA Expression
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To validate the changes of treatment-related tsRNAs detected by RNA-seq, all tsRNAs were selected and their expression was examined by qPCR. Firstly, total RNA was reverse-transcribed into cDNA using rtStar™ First-Strand cDNA Synthesis Kit (3' and 5' adaptor; Arraystar) according to the manufacturer's protocol. Then, qPCR amplification was performed using ViiA 7 Real-time PCR System (Applied Biosystems) and 2×PCR master mix (Arraystar). The cycling conditions were as follows: incubation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 60 s and 95 °C for 15 s. The relative tsRNA expression levels were calculated using the 2-ΔΔCt method and were normalized to U6, as an endogenous. The specific primers for each gene were listed in Table 1 . All reactions were performed in triplicate.
Furthermore, the target mRNAs of treatment- related tsRNAs were validated by qPCR as well. One target of each tsRNA was randomly selected. The qPCR protocols were described as above and β-Actin was used as internal controls to normalize the data.
Furthermore, the target mRNAs of treatment- related tsRNAs were validated by qPCR as well. One target of each tsRNA was randomly selected. The qPCR protocols were described as above and β-Actin was used as internal controls to normalize the data.
9
Validating tsRNA Expression by qRT-PCR
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To validate the tsRNA sequencing results, the expression of selected tsRNAs was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA isolated from PBMCs was pretreated with the rtStar tRF&tiRNA Pretreatment Kit (Arraystar, Rockville, MD), and the cDNA was synthesized using the rtStar First-Strand cDNA Synthesis Kit (3’ and 5’ adaptor; Arraystar, Rockville, MD) according to the manufacturer's protocol. The primer pairs of each gene are listed in Table 1 (KangChen Bio-tech, Shanghai, China). qRT-PCR was performed using a ViiA 7 Real-time PCR System (Applied Biosystems, Foster City, CA) with 2 × PCR Master Mix (Arraystar, Rockville, MD); thermal cycling conditions were 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 60 seconds. The relative expression level of tsRNAs was calculated using the comparative Ct (2−ΔΔCt) method and normalized to U6.
10
Quantitative Analysis of tiRNAs and mRNAs
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Total RNA was extracted using TRIzol (Invitrogen) from vasculature, cells, or plasma according to the manufacturer’s protocol. tiRNA reverse transcription, cDNA synthesis, and quantitative real-time PCR (qRT-PCR) were performed as previously described (34 (link)). Briefly, the rtStar™ tRF and tiRNA Pretreatment Kit (Cat# AS-FS-005, Arraystar, Rockville, MD, USA) was used to remove RNA modifications, and the rtStar™ First-Strand cDNA Synthesis Kit (3′ and 5′ adaptor) (Cat# AS-FS-003, Arraystar, Rockville, MD, USA) was used to synthesize cDNA. For mRNA reverse transcription, cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix (TOYOBO, Japan). For qRT-PCR, THUNDERBIRD® Next SYBR qPCR Mix (TOYOBO, Japan) was used according to the manufacturer’s instructions, and the procedure was performed on a QuantStudio™ 5 Real-time PCR System (Applied Biosystems, USA). U6 and glyceraldehyde phosphate dehydrogenase (GAPDH) were regarded as the reference for tiRNA and mRNA, respectively. The expression levels were analyzed by the ΔCq method, and 2–ΔΔCq was used to calculate the relative expression. Primers are listed in Supplementary Table 1 .
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