RNA extraction was carried out using RNAiso (Takara) following the instructions of the manufacturer. Extracted RNAs were treated with DNaseI (Takara) to remove contaminating genomic DNA, and further purified using RNAiso. Total RNA was further purified by poly-A selection in some experiments. Total RNA and poly-A selected RNA were analyzed by Northern blotting using DIG-labeled cRNA probe according to the manufacturers’ instructions (Roche). Reverse transcription (RT) was performed with 2 µg of total RNA, oligo dT (20 mer) and ReverTra Ace (TOYOBO). The resulting cDNA was subjected to qPCR in a Roche LightCycler with SYBR Premix Ex Taq (Takara). Primers are listed in Text S1 .
Rnaiso
Manufactured by Takara Bio
Sourced in Japan, China, United States
RNAiso is a total RNA extraction reagent. It is designed for the isolation of high-quality total RNA from a variety of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (32 mentions)
Sybr premix ex taq 2, by Takara Bio (24 mentions)
Primescript rt master mix, by Takara Bio (16 mentions)
Sybr premix ex taq, by Takara Bio (15 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (12 mentions)
M mlv reverse transcriptase, by Promega (10 mentions)
Lightcycler 480 system, by Roche (8 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (8 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (8 mentions)
Sybr premix ex taqtm, by Takara Bio (7 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Revertra ace, by Toyobo (6 mentions)
Sybr green qpcr mix, by Bio-Rad (6 mentions)
Thunderbird sybr qpcr mix, by Toyobo (6 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (6 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (5 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (5 mentions)
Tb green premix ex taq 2, by Takara Bio (5 mentions)
Primescript first strand cdna synthesis kit, by Takara Bio (5 mentions)
278 protocols using rnaiso
1
Comprehensive RNA Extraction and Analysis
2
Quantitative Real-time RT-PCR Analysis
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Real-time reverse transcription-PCR was performed as previously described (Akatsu et al., 2015) . In brief, each region of the left hemisphere was homogenized in RNAiso (Takara Bio, Shiga, Japan) and total RNA was isolated using RNAiso (Takara Bio), according to the manufacturer's instructions. The isolated 1 µg of total RNA was reverse-transcribed using the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany). PCR was performed using the Thermal Cycler Dice Real Time TP800 System (Takara Bio) with SYBR Premix Ex Taq II (Takara Bio). The thermal cycling conditions comprised an initial step at 95℃ for 10 sec, followed by 50 cycles at 95℃ for 5 sec and at 60℃ for 30 sec. Table 1 lists the primer sequences. The BDNF, GluR1, and GluR2 genes have splicing variants. The primers we used are designed to capture all the splicing variants. The 5-HT 1A receptor, GABA A receptor α2 and α3 subunits, and 18S rRNA genes do not have any splicing variants. The sizes of the PCR products were verified using agarose gel electrophoresis. Quantitation was performed using the crossing point method. The data was normalized to 18S rRNA.
3
Silencing STAB1 in Leukemia Cell Lines
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KG1a, NB4, THP-1, K562, MOML13, HL60, and U937 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL ampicillin, and incubated in a humidified atmosphere containing 5% CO2 and 95% air at 37°C. For transfection, the cultured cell lines were seeded in six-well plates and transfected with 100 nmol/L STAB1 siRNAs or control oligonucleotides (NC) (RiboBio, Guangzhou, China). The sequences for si-STAB1-1, si-STAB1-2, and si-STAB1-3 were 5′-GGATCGTCTTCTACAACCA-3′, 5′-AGATCACCGTCACCTTTAA-3′, and 5′-GGAACAATGGTCACTTGTA-3′, respectively.
After 48 h of transfection, total RNA was extracted from KG1a cells using RNAiso (Takara) following the manufacturer’s protocol, and 1 μg of the RNA was reverse transcribed using PrimeScript RT Master Mix (Takara). Quantitative real-time PCR (real-time qPCR) of STAB1 was performed using the cDNA as a template in the presence of SYBR Premix Ex Taq (Tli RNaseH Plus; Takara). The levels of mRNA were normalized to the level of actin.
After 48 h of transfection, total RNA was extracted from KG1a cells using RNAiso (Takara) following the manufacturer’s protocol, and 1 μg of the RNA was reverse transcribed using PrimeScript RT Master Mix (Takara). Quantitative real-time PCR (real-time qPCR) of STAB1 was performed using the cDNA as a template in the presence of SYBR Premix Ex Taq (Tli RNaseH Plus; Takara). The levels of mRNA were normalized to the level of actin.
4
Quantitative Analysis of Gene Expression
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Each group was added RNAiso (TaKaRa, Japan) to extract the total RNA. The PrimeScript RT reagent kit (TaKaRa, Japan) was used to reverse RNA to cDNA. Then, the SYBR Premix Ex Taq II kit (TaKaRa, Japan) was used for RT-PCR. The CT values of Piezo1, Kif18A, β-tubulin, and GAPDH genes were obtained by the FTC-2000 system (Applied Biosystems, China), and the parameters were as follows: predenaturation: 95°C for 30 seconds, 1 cycle; PCR: 95°C for 5 seconds, 60°C for 30 seconds, and 72°C for 30 seconds for 40 cycles. The relative expression of the target gene was calculated by 2−ΔΔCt. GAPDH, Piezo1, Kif18A, and β-tubulin primers were produced by Shanghai Bioengineering Company (Table 2 ).
5
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA from tested tissues was extracted by RNAiso (TaKaRa). According to the manufacturer’s instructions, the first-strand complementary DNA (cDNA) synthesis was carried out with 1 μg total RNA by the PrimeScript™ RT reagent Kit (TaKaRa). The qRT-PCR was performed on the ABI 7500 real-time PCR machine with a total 20 μL reaction, which contained SYBR (TaKaRa) 10 μL, 10 mM forward and reverse primer 0.4 μL respectively, and diluted cDNA 0.1 μL. The ACTIN gene was adopted as the internal control for normalization (Cao et al., 2018 (link)). The qRT-PCR results were obtained from three biological replicates. The relative expression levels were summaried based on the 2−ΔΔct method (Livak and Schmittgen, 2001 (link)).
6
Quantitative Gene Expression Analysis
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Total RNA was extracted using RNAiso (TaKaRa, Dalian, China) and reverse transcribed into cDNA using the Prime Script RT MasterMix Kit (TaKaRa). Real-time PCR was conducted using SYBR MasterMix (Thermo) according to the manufacturer's instructions. mRNA expression of target genes was normalized to GAPDH using the 2-ΔΔct method. The primer sequences were as follows: Ajuba for 5′-GATGCGGGAGCCAGAGG-3′, rev 5′-CACAAGAGCAGCAAACAAAGC-3′; Survivin for 5′-ACCGCATCTCTACATTCAAG-3′, rev 5′-CAAGTCTGGCTCGTTCTC-3′; GLUT3 for 5′-CCTTTGGCACTCTCAACCAGC-3′, rev 5′-AACCCAGTAGCAGCGGCCAT-3′; and GADPH for 5′-GAAATCCCATCACCATCTTCCAG-3′, rev 5′-GAGCCCCAGCCTTCTCCAT-3′.
7
Transcriptional Analysis of savRS in S. aureus
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Overnight cultures of S. aureus were diluted 1:100 in TSB and grown at 37°C. The cells were collected at the indicated cell density and processed with 1 ml RNAiso (TaKaRa) in combination with 0.1-mm-diameter zirconia-silica beads in a FastPrep-24 automated system (MP Biomedicals). The residual DNA was removed with RNase-free DNase I (TaKaRa). Transcription analysis of savRS was performed by reverse transcription-PCR using Moloney murine leukemia virus reverse transcriptase (TaKaRa) with primer RT-savS, and the PCR products were analyzed with the primers savS-F/savS-R and savS-F/savR-R. Reverse transcription for qRT-PCR analysis was performed using a PrimeScript first-strand cDNA synthesis kit (TaKaRa) with random primers. Real-time PCR was carried out with SYBR premix Ex Taq (TaKaRa) using a StepOne real-time PCR system (Applied Biosystems). The quantity of cDNA measured was normalized to the hu cDNA abundance. The primers used in this study are listed in Table 3 .
8
Quantifying GOLPH3 Gene Expression in Cell Lines
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For each cell line, the total RNA was extracted using RNAiso (Takara, Dalian, China), and reverse transcription was performed using RevertAid Reverse Transcriptase (Fermentas, Glen Burnie, MD), according to the manufacturer's instructions. The cDNA was used to amplify the GOLPH3 gene using PCR with the following primers: GOLPH3 forward, 5'-ACATCCCCTCACCAATAACAAC-3', and GOLPH3 reverse, 5'-TAGCCAAATCATACTGCTCGTC-3'. The QPCR reactions were performed in triplicate in a 15 μl reaction volume containing 7.5 μl of 2×SYBR mix (Takara), 0.2 μl of each primer (10 μM), and 0.3 μl cDNA. The conditions were as follows: 94°C for 2 min, followed by 40 cycles at 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec. The expression values of GOLPH3 were normalized to the endogenous reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
9
Quantitative PCR Analysis of Gene Expression
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Total RNA was extracted from the cells using RNAiso (Takara, 9109). First-strand cDNA synthesis was performed using the PrimeScriptTM RT reagent kit (Takara, RR037A). The obtained cDNAs were analyzed through quantitative PCR (qPCR) with SYBR® Premix Ex TaqTM (Takara, RR420A) using an Agilent Stratagene Mx3000P instrument. The primer sequences used in this study for qPCR are listed in Supplementary Table 5 .
10
Quantitative RNA Expression Analysis
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Total RNA was extracted from cells or tissues by the phenol chloroform method using RNAiso (Takara Bio, Inc). Using the PrimeScript RT reagent kit (Takara Bio, Inc), cDNA was synthesized from 1 μg of total RNA after removal of genomic DNA. Random primers were used for reverse transcription reactions other than strand specific. qPCR was performed using Thermal Cycler Dice Real Time System III (Takara Bio, Inc) according to recommended cycling parameters using 1 μl cDNA diluted fivefold with EASY Dilution (Takara Bio, Inc) and TB Green Premix Ex Taq TMII (Takara Bio, Inc). Gene expression levels were normalized to GAPDH by the ΔΔCT method. Primer information for chain-specific RT and primer walking, and for other qRT–PCR, is detailed in Tables S1 and S2 , respectively.
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