Total RNA was extracted using ISOGEN II (Wako) according to the manufacturer's instructions. For qRT-PCR analysis, cDNAs were synthesized using a SuperScript II cDNA synthesis kit (Invitrogen). Real-time PCR amplifications were performed in 96-well optical reaction plates with Power SYBR Green PCR Master Mix (Applied Biosystems). The relative expression values of each gene were determined by normalization to GAPDH expression for each sample. Primer sequences: p53-Fw (5′-TCAACAAGATGTTTTGCCAACTG-3′), p53-Rv (5′-ATGTGCTGTGACTGCTTGTAGATG-3′), p21-Fw (5′-TCAGGGTCGAAAACGGCG-3′), p21-Rv (5′-AAGATCAGCCGGCGTTTGGA-3′), Fbxo22-Fw (5′-CTCACTGAAGTAGGTCTTTTAG-3′), Fbxo22-Rv (5′-CCAGCCAAGATGATATTCATATC-3′), Hdm2-Fw (5′-ACCTCACAGATTCCAGCTTCG-3′), Hdm2-Rv (5′-TTTCATAGTATAAGTGTCTTTTT-3′), GAPDH-Fw (5′-GAGTCAACGGATTTGGTC GT-3′), GAPDH-Rv (5′-TTGATTTTGGAGGGATCTCG-3′), IL-6-Fw (5′-CCAGGAGCCCAGCTATGAAC-3′), IL-6-Rv (5′-CCCAGGGAGAAGGCAACTG-3′), IL-8-Fw (5′-AAGGAAAACTGGGTGCAGAG-3′), IL-8-Rv (5′-ATTGCATCTGGCAACCCTAC-3′)
Isogen 2
Manufactured by Fujifilm
Sourced in Japan
The ISOGEN II is a high-performance laboratory instrument designed for DNA/RNA isolation and purification. It utilizes a proprietary extraction method to efficiently extract and purify nucleic acids from a variety of sample types. The ISOGEN II is a versatile and reliable tool for researchers and laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (3 mentions)
Revertra ace, by Toyobo (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Superscript 2 cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Thunderbird sybr qpcr mix, by Toyobo (2 mentions)
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Primetime gene expression master mix, by Integrated DNA Technologies (1 mentions)
Luna qpcr master mix, by New England Biolabs (1 mentions)
Lightcycler 480 2 system, by Roche (1 mentions)
Revertra ace qpcr rt master mix with gdna remover, by Toyobo (1 mentions)
Power syber green pcr master mix, by Thermo Fisher Scientific (1 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Abi prism 7000, by PerkinElmer (1 mentions)
Kod sybr qpcr mix, by Toyobo (1 mentions)
Abi prism 7300, by Thermo Fisher Scientific (1 mentions)
Novaseq platform, by Illumina (1 mentions)
23 protocols using isogen 2
1
Quantitative Real-Time PCR Analysis of Gene Expression
2
Quantitative Real-Time PCR Analysis
3
Quantification of Bacterial Gene Expression by qRT-PCR
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Total RNA was isolated by ISOGEN II (Wako), and the concentration was determined by measuring the A260 value. Subsequently, 0.5 μg of RNA from each sample was reverse‐transcribed by using PrimeScript™ II 1st strand cDNA Synthesis Kit (Takara) according to the manufacturer's protocol. To obtain standard curves for the hmpA, fur, and tufA genes, genomic DNA from EHEC EDL933 was 10‐fold serially diluted from 1.0 × 105 to 0.1 cfu/2 μl and amplified in the 7,300 Real‐Time PCR System (Applied Biosystems, CA, USA) with the primers (P16 ‐ P17), (P1243 – P1244) or (P890 ‐ P891) using the Power SYBER Green PCR Master Mix (Applied Biosystems) according to the manufacturer's protocol, respectively. Serial dilutions of cDNA were amplified in the 7300 Real‐Time PCR System under the same conditions as for the standard curves with the primers (P16 ‐ P17), (P1243 – P1244), or (P890 ‐ P891). The levels of hmpA, fur, and tufA mRNA were quantified by noting the fluorescence crossing point of the samples on the corresponding standard curve, and the results are presented as the ratio among the expression levels of hmpA mRNA, fur mRNA, and tufA mRNA.
4
Real-Time PCR Analysis of Bcl-xL Expression
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Total RNA from HeLa cells (2 × 105 cells) was extracted by ISOGEN II (WAKO) as described in the instruction manual. First-strand cDNA synthesis was performed with a PrimeScript™ II 1st strand cDNA Synthesis Kit (Takara Bio). Real-time quantitative PCR (qPCR) analysis was conducted using KOD SYBR qPCR Mix (TOYOBO) with the fluorescent dye SYBR Green and a ABI Prism 7000 (PerkinElmer Life Sciences) for detection. Primer pairs for the Bcl-xL gene PCR were synthesized according to the previous report59 (link). The PCR protocol was performed as described in the instruction manual. The reaction mixture was activated at 98 °C for 2 min of 1 cycle, followed by denaturation for 10 s at 98 °C, annealing for 10 secs at 60 °C and extension for 30 s at 68 °C, for 45 cycles. The dissociation curve for each sample was analyzed to verify the specificity of each reaction. The relative mRNA expression levels of Bcl-xL genes were determined by the delta-delta Ct method and normalized to β-actin expression. Specific primers were as follows: sense; 5′-TTCTACAATGAGCTGCGTGTG-3′ and antisense 5′-GGGGTGTTGAAGGTCTCAAA-3′.
5
Quantitative Analysis of Cx43 mRNA Expression
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Total RNA from AZ-521 cells (2×105 cells) was extracted by ISOGEN II (Wako) as described in the instruction manual. First-strand cDNA synthesis was performed with a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio). Real-time quantitative PCR (qPCR) analysis was conducted with the fluorescent dye SYBR Green methodology using Power SYBR Green PCR Master Mix (ABI) and ABI Prism 7300 (Applied Biosystems Inc, Carlsbad, CA, USA). Primer pairs for the Cx43 and GAPDH genes were obtained from Takara. The PCR protocol was performed as described in the instruction manual: the reaction mix containing AmpliTaq Gold DNA polymerase was incubated for 10 min at 95 °C, followed by denaturation for 15 s at 95 °C with annealing and extension for 1 min at 60 °C, for 45 cycles. The dissociation curve for each sample was analyzed to verify the specificity of each reaction. The relative mRNA expression levels of Cx43 genes were determined by the delta-delta Ct method and normalized to GAPDH expression.
6
RNA-seq of Luc and Siwi siRNA Treated BmN4 Cells
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BmN4 cells treated with Luc (control) or Siwi siRNA were used for RNA sequencing. Total RNAs were isolated using ISOGEN II (FUJIFILM Wako Pure Chemical) and DNase (Life Technologies). RNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) with the Ribo-Zero Plus rRNA Depletion Kit (Human/Mouse/Rat) (Illumina) and sequenced on the NovaSeq platform (Illumina) to obtain paired-end reads, resulting in 77,614,516 and 68,702,400 reads for Luc KD and 81,135,072 and 77,184,922 reads for Siwi KD (Replicates 1 and 2, respectively).
7
Quantifying Gene Expression in Tissue Samples
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After collection, tissue samples were immediately immersed in RNAlater RNA stabilization reagent (Qiagen, Holden, Germany) and stored until use. Total RNA was extracted with ISOGEN II (Wako Pure Chemical Industries). After purification, complementary DNA (cDNA) was synthesized with a high-capacity cDNA reverse transcription kit (Life Technologies, Carlsbad, CA, USA). After cDNA synthesis, gene expressions of c-kit, CK5, AQP5, α-amylase and GAPDH were analyzed using the reverse transcription polymerase chain reaction (RT-PCR) and quantified with real-time PCR. A reaction solution was prepared with Fast SYBR® Green Master Mix (Applied Biosystems, Foster City, CA, USA) using appropriate primers (Table 2 ). Real-time RT-PCR was performed using StepOnePlusTM (Applied Biosystems). After initial heating at 95°C for 20 sec, PCR conditions were set at 95°C for 3 sec and 60°C for 30 sec for 40 cycles each. The levels of RNA expression in the 2M lig group were considered as baseline and were analyzed using the ΔΔCT method. Paired two-way ANOVA and Bonferroni multiple comparisons were performed for statistical analysis (Ekuseru-Toukei 2015). A p value < 0.05 was considered statistically significant.
8
Total RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted from cells with ISOGENII (311–07361; Wako) or the RNeasy Mini Kit (Qiagen) and was subjected to cDNA synthesis with random hexamer primers and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen) according to the manufacturer’s protocols. RT-qPCR was performed with FastStart Universal SYBR (Roche) and a Thermal Cycler Dice Real Time System II (TP900; Takara Bio). The PCR primers that were used are listed in S2 Table . 18S rRNA was used for normalization.
9
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from tissues and cells using ISOGEN II (FUJIFILM Wako Pure
Chemical Corp.) following the manufacturer’s instructions. For the reverse
transcription-polymerase chain reaction (RT-PCR), first-strand cDNA was synthesized using
total RNA (1 µg) with random hexamers and ReverTra Ace reverse
transcriptase (TOYOBO Co., Ltd., Osaka, Japan). The cDNA template was amplified using
BIOTAQTM HS DNA Polymerase (Bioline Ltd.; London, UK) and specific primers
for dog SF-1 and dog glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). All PCR experiments were performed
under the following thermocycling conditions: 95°C for 10 min; 30 cycles of 95°C for 30
sec, 60°C for 30 sec, and 72°C for 1 min, with a final extension at 72°C for 10 min.
Quantitative real-time PCR (qPCR) was performed using SYBR® Green PCR master
mix (Applied Biosystems, Woburn, MA, USA). Data were normalized to GAPDHexpression. Gene expression levels are presented as the fold-change in expression, which
was calculated using the Pfaffl method [22 (link)]. The
sequences of the primers used in this study are summarized inTable 1 .
Chemical Corp.) following the manufacturer’s instructions. For the reverse
transcription-polymerase chain reaction (RT-PCR), first-strand cDNA was synthesized using
total RNA (1 µg) with random hexamers and ReverTra Ace reverse
transcriptase (TOYOBO Co., Ltd., Osaka, Japan). The cDNA template was amplified using
BIOTAQTM HS DNA Polymerase (Bioline Ltd.; London, UK) and specific primers
for dog SF-1 and dog glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). All PCR experiments were performed
under the following thermocycling conditions: 95°C for 10 min; 30 cycles of 95°C for 30
sec, 60°C for 30 sec, and 72°C for 1 min, with a final extension at 72°C for 10 min.
Quantitative real-time PCR (qPCR) was performed using SYBR® Green PCR master
mix (Applied Biosystems, Woburn, MA, USA). Data were normalized to GAPDHexpression. Gene expression levels are presented as the fold-change in expression, which
was calculated using the Pfaffl method [22 (link)]. The
sequences of the primers used in this study are summarized in
10
Quantifying Dicer and miRNA Expression
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Total RNA was extracted with Isogen II (Wako Chemicals, Osaka, Japan), and 1 µg of total RNA was converted to cDNA with ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan). Real-time PCR was performed with Thunderbird® SYBR® qPCR Mix (Toyobo), with gene-specific primers. The following primers were used for real-time PCR: Dicer, 5′-CACACGCCTCCTACCACTACAACAC-3′ and 5′-CCGTGGGTCTTCATAAAGGT-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5′-AAATGGTGAAGGTCGGTGTG-3′ and 5′-TGAAGGGGTCGTTGATGG-3′. For the real-time PCR analysis of mature miRNA expression, the TaqMan® MicroRNA Reverse Transcription Kit and TaqMan® MicroRNA Assays (Applied Biosystems, Foster City, CA, USA) were used, according to the manufacturer’s protocols [38 (link),39 (link)].
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