Total RNA was isolated from viral infected K562 cells using the RNAiso Plus RNA extraction reagent (TaKaRa, Japan). After cDNA synthesis, the cDNA was quantified and used for mRNA expression analysis. The PCR primers are summarized in Supplementary Table S1 . Dissociation curves were examined after each PCR run to verify the amplification of a single product of the appropriate length. The mean threshold cycle (CT) and standard error values were calculated from individual CT values obtained from triplicate reactions per stage. The normalized mean CT value was estimated as ΔCT by subtracting the mean CT of GAPDH. ΔΔCT values were calculated as the difference between the control ΔCT and the values obtained for each sample. The n-fold changes in gene expression relative to an untreated control were calculated as 2-ΔΔCT.
Rnaiso plus rna extraction reagent
Manufactured by Takara Bio
Sourced in Japan
RNAiso Plus is a reagent for the isolation of total RNA from various biological samples. It is designed to effectively extract and purify RNA using a single-step liquid-liquid extraction method.
Automatically generated - may contain errors
Lab products found in correlation
Reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Abi prism 7900ht real time pcr machine, by Thermo Fisher Scientific (1 mentions)
Sybr green qpcr master mix, by Takara Bio (1 mentions)
Abi prism 7500 system, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
3 protocols using rnaiso plus rna extraction reagent
1
Quantitative RT-PCR Analysis of Viral Gene Expression
2
RNA Extraction and qRT-PCR Analysis
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Extraction of RNA (total RNA) was performed on each sample by utilizing an RNAiso Plus RNA extraction reagent (Takara, Tokyo, Japan) following the specific manufacture’s protocol. Then, the isolated RNA from each sample was reverse transcribed into cDNA templates by making use of a iScript™ cDNA Synthesis Kit (Life Science Research, Hercules, CA) along with a Reverse Transcription kit (Thermo Fisher Scientific, MA) following assay protocols provided by manufacturers along with the assay kit. Then, qRT-PCR was performed using a Fast Start Universal SYBR Green Master Mix assay kit (Roche, Basel, Switzerland) in accordance with the specific assay protocol provided by the assay kit manufacturer. The real-time PCR reaction was done on an ABI Prism 7900HT real-time PCR machine (Applied Biosystems, Foster City, CA) using U6 as well as GAPDH as the internal control for the normalization of measured relative expression of target genes, i.e., circRNA_FOXO3 (Forward: 5’-GTGGGGAACTTCACTGGTGCTAAG-3’; Reverse: 5’-GGGTTGATGATCCACCAAGAGCTCTT-3), miR-23a (Forward: 5’-TTCCTGGGGATGGGATT-3’; Reverse: 5’-GAACATGTCTGCGTATCTC-3’), IL-6 (Forward: 5’-AGACAGCCACTCACCTCTTCAG-3’; Reverse: 5’-TTCTGCCAGTGCCTCTTTGCTG-3’), IL-1β (Forward: 5’-CCACAGACCTTCCAGGAGAATG-3’; Reverse: 5’-GTGCAGTTCAGTGATCGTACAGG-3’), and TNF-α (Forward: 5’-CTCTTCTGCCTGCTGCACTTTG-3’; Reverse: 5’-ATGGGCTACAGGCTTGTCACTC-3’).
3
Dose-Dependent Effect of Sarsasapogenin on Osteoclast Gene Expression
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Osteoclasts were collected from BMMs that cultured with M-CSF (25 ng/mL) and RANKL (100 ng/mL) under various concentrations of sarsasapogenin (0, 1, 2, or 4 μM) for the dose-dependent effect of sarsasapogenin. RNAiso Plus RNA Extraction Reagent (Takara Bio, Japan) was used to isolate the total RNA from cells as previously described. cDNA (Complementary DNA) was synthesized using 1 μg extracted RNA and Prime Script RT Master Mix (Takara Bio). The cDNA was used as a template for qPCR using SYBR Green qPCR Master Mix (Takara Bio) on an ABI Prism 7500 System (Applied Biosystems, CA, USA) under the following conditions: denaturation at 95°C for 10 min; 95°C for 10 s, 60°C for 20 s, 72°C for 20 s, 40 cycles in total; 72°C for 90 s as the final extension. Specific primers of mouse are shown in Table 1 .
Mouse Primers for qPCR
| Gene | Forward Primer | Reverse Primer |
|---|---|---|
| β-Actin | 5ʹ-AGC CAT GTA CGT AGC CAT CC-3ʹ | 5ʹ-CTC TCA GCA GTG GTG GTG AA-3ʹ |
| TRAP | 5ʹ -TCC TGG CTC AAA AAG CAG TT-3ʹ | 5ʹ-ACA TAG CCC ACA CCG TTC TC-3ʹ |
| CTSK | 5ʹ-CTT CCA ATA CGT GCA GCA GA-3ʹ | 5ʹ-TCT TCA GGG CTT TCT CGT TC-3ʹ |
| NFATc1 | 5ʹ-CAG CTG CCG TCG CAC TCT GGT C-3ʹ | 5ʹ-CCC GGC TGC CTT CCG TCT CAT A-3ʹ |
| ATP6V0d2 | 5ʹ-AAG CCT TTG TTT GAC GCT GT-3ʹ | 5ʹ-TTC GAT GCC TCT GTG AGA TG-3ʹ |
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