Briefly, cells were extracted using trizol reagent invitrogen, usa and cdnas were synthesized by primescript rt reagent kit with gdna eraser, according to the manufacturers recommendation. Maxima first strand cdna synthesis kit for rtqpcr with dsdnase thermo fisher scientific. Viral genomic dna or rna were extracted from plasma, lung, and spleen samples. Primescript rt reagent kit perfect real time rr037a. An actin gene as the internal control for gene expression. Second, using the primescript rt reagent kit with gdna eraser, genomic. Then mrnas were reverse transcribed with primescript rt reagent kit with genomic deoxyribonucleic acid gdna eraser takara, dalian. The overexpression of three cytochrome p450 genes cyp6cy14. Research paper trim59 is a novel marker of poor prognosis. Realtime qpcr was performed on cfx96 biolab, with endogenous control actb. Firststand cdna was synthesized using the primescript rt reagent kit with gdna eraser takara, dalian, china following the manufacturers instructions. Primescript rt reagent kit with gdna eraser takara. The primescript rtpcr kit is a twostep rtpcr kit featuring primescript rt, an rnase h minus mmlv reverse transcriptase enzyme that provides robust reverse transcription of virtually any rna template.
The concentration and purity of all total rna samples were veri. Six important factors for successful reverse transcription. Primescript rt reagent kit perfect real time primestar gxl dna polymerase. Sirius red staining connective tissue staining kit was purchased from abcam ab150681, cambridge, uk. Primescript rtase is a modified, recombinant mmlv moloney murine leukemia. Maxima first strand cdna synthesis kit for rtqpcr with dsdnase thermo fisher scientific d. Primescript rt reagent kit with gdna eraser perfect real time cat. Reverse transcription reactions can be completed in as few as 15 minutes. This rtpcr kit is conveniently configured to include all reagents needed for twostep endpoint rtpcr, including primers, primescript rtase, and pcr polymerase enzyme. Quantitative rtpcr total rna from pmnmdscs and mmdscs was extracted usingthetrizolreagentinvitrogenlifetechnologies,andrna using the total of 5 mg cdna was synthesized from singlestrand primescript rt reagent kit with gdna eraser takara. Rr420a, takara, shiga, japan was used to measure the gene expression patterns. Triptolide decreases expression of latencyassociated.
Realtime pcr was carried out with a biorad cfx connect real. Evaluation of reference genes for gene expression studies. The realtime rtpcr kit contains a 5x master mix reagent that includes all reagents required for cdna synthesis primescript rtase, rnase inhibitor, random 6mers, oligo dt primer, dntps, and reaction buffer. Cloning gp and gs genes primers were designed in vector nti suite 7 software using cdna as the template and according to the sequences of gp and gs obtained from transcriptome sequencing. The primescript rt reagent kit with gdna eraser and sybr premix ex taq ii tli rnaseh plus kit were from takara clontech kyoto, japan. Annexin vfluoresceine isothiocyanate fitcpropidium iodide pi apoptosis detection kit, cell cycle detection kit, and bicinchoninic acid bca protein assay kit were obtained from. D, primescript rt reagent kit with gdna eraser takara. A sybr premix ex taq ii kit takara, shiga, jpn was used for gene amplification by a qrt. Total rna was extracted using rneasy plus mini kit 74104. Metformininduced reduction of cd39 and cd73 blocks. Transcription factors responding to pb stress in maize.
The expression of the selected lncrnas nonhsat9215 and nonhsat219 was analyzed using qrtpcr with a sybr green pcr kit takara and realtime pcr system roche, lightcycler 96. Primescript rtpcr kit from takara bio selectscience. Primescript rt reagent kit perfect real time from takara bio. This realtime rtpcr kit combines high yield with high sensitivity pcr. Aliquots of 2 mg of mrna were reversetranscribed using a primescript rt reagent kit with the gdna eraser kit according to the manufacturers instructions takara biotechnology, china. H19 lncrna identified as a master regulator of genes that. Drr037a primescript rtreagent kit perfect real time 200 rna. Realtime reverse transcription quantitative polymerase chain reaction rtqpcr for cdna synthesis, 2. Primescript rt reagent kit perfect real time 200 takaracode. Finally, the mrna expression of the abcb11 gene in the pis patients was detected by rtqpcr using sybr premix ex taq ii takara bio, inc. Dna removal and reverse transcription were performed with the primescript rt reagent kit with gdna eraser takara bio, inc. L of rna was taken and reverse transcribed into cdna using the primescript rt reagent kit with gdna eraser.
The genomic dna sequence of mietr1b was 4 116 bp in length, having a 3 305. Sybr greenbased qpcr was then performed on an abi viia 7 realtime. Rna extracted from small intestine tissue was reversetranscribed into cdna according to primescript rt reagent kit with gdna eraser instruction manual, and the objective band was observed after pcr amplification. Agronomy 2018, 8, 186 4 of with a primescripttm rt reagent kit with a gdna eraser perfect real time, code no. Transcriptome sequencing reveals key pathways and genes. Rnaseq identifies determinants of oxaliplatin sensitivity. Primescript rt reagent kit with gdna eraser is a reversetranscription kit for realtime rtpcr rt qpcr that includes a genomic dna elimination reaction. Then the total rnas were reversely transcribed to cdnas using primescript rt reagent kit with gdna eraser takara. Primescript rt reagent kit with gdna eraser perfect real time. Realtime polymerase chain reaction assessment a primescript rt reagent kit with gdna eraser takara, otsu, japan was use to reverse transcribe equal amounts of dnafree rna 5 mg from different tissues into cdna. Isolation and expression analysis of the ethylene receptor gene.
Growth hormone promotes human endometrial glandular cells. D, primescript rt reagent kit with gdna eraser takara, indicating a significant amount of residual gdna. The mrna expression was analyzed by qrtpcr, with the primer sequences shown in table 1. Total rna above isolated was synthesized to cdna using primescript rt reagent kit with gdna eraser takara, rr074a for rtpcr with mixture of oligodt and random primer 9 mer. Or use a onestep rt rtpcr primescript kit and set your reverse. Primescript rtase is an mmlv reverse transcriptase and enables fulllength cdna synthesis. Pcr rtqpcr that includes a genomic dna elimination reaction. The primescript rt reagent kit perfect real time is a reverse transcription kit for qpcr that provides high yields and high sensitivity. The primers used for qpcr validation were list in supplementary table 1. Rna extraction and realtime pcr total rna was extracted with trizol ambion from all four tissues, rna concentrations were measured by nanodrop, and the.
Total rna was transcribed using the primescript rt reagent kit with the gdna eraser takara, dalian, china. A pair of degenerate primers gstn and gstc were designed according to the conserved sequences of class i and class iii gst genes from other lepidopteran species. Cell counting kit8 cck8 was from dojindo kumamoto, japan, and 4well culture inserts were from ibidi cat. Total rna of the collected samples was extracted following the above mentioned method, and then was reversetranscribed using the primescript rt reagent kit. The cdna was synthesized from 1 g rna using primescript rt reagent kit with gdna eraser takara, kyoto, japan. Insights into grapevine defense response against drought. Primescript rt reagent kit with gdna eraser perfect real. The cdna synthesis was conducted using primescript rt reagent kit with gdna eraser, and the synthesized cdna was kept at.
Takara bio realtime pcr and rtpcr reagents designed. The expression of lncrna neat1 in human tuberculosis and. Amplification was detected from other kits b, quantitect reverse transcription kit qiagen. Rtpcr was performed by applied biosystems 7900ht fast realtime pcr system thermo, usa. Identification and validation of key genes with prognostic. Analysis of long noncoding rna expression in hemophilia a. Primescript rt reagent kit with gdna eraser is a reversetranscription kit for realtime rt. This rtpcr kit is conveniently configured to include all reagents needed for twostep endpoint rtpcr, including primers. Table s2 were designed using the software primer premier 5.
Contribution of tissue transglutaminase to the severity of. All detection steps and reaction conditions were set according to the instruction provided with the kit. Trizol was obtained from life technologies waltham, usa. Modeling of early neural development in vitro by direct. With the primescript rt reagent kit with gdna eraser perfect real time, genomic dna contamination is eliminated. Avoid genomic dna contamination during cdna synthesis and rtqpcr, primescript rt reagent kit with gdna eraser perfect real time cat. Total rna was isolated from the huvecs using the rneasy mini kit qiagen, valencia, ca and the quantity was measured using a spectrophotometer, as previously described. C, maxima first strand cdna synthesis kit for rtqpcr with dsdnase thermo fisher scientific. Quantitative realtime pcr was conducted using a tb green premix ex taq ii tli rnaseh plus takara on a biorad cfx96 system according to the manufacturers instructions.
Qiagen, hilden, germany and reverse transcribed using primescript rt reagent kit with gdna eraser rr047a. Onestep tb green primescript rtpcr kit ii perfect real time cat. Genomewide identification and expression analysis of the. The primescript rt reagent kit is a reverse transcription kit for qpcr. Quantitative realtime pcr rtpcr total rnas were extracted from the human ovarian tissues and cell lines using trizol reagent invitrogen. The primescript rt reagent kit perfect real time includes primescript reverse transcriptase for strong stranddisplacement activity and efficient synthesis of cdna for realtime pcr. Primescript rt reagent kit with gdna eraser takara fig. Functional analysis of the correlation between abcb11 gene. Effects of high glucose on the expression of lama1 and. Genomic dna elimination reactions were performed in a volume of 10. The activation of nrf2 and its downstream regulated genes. Rtpcr amplification of bax, bcl2 and betaactin gene. This reverse transcription kit enables fast cdna synthesis for realtime rtpcr qpcr and includes a rapid step that eliminates genomic dna gdna contamination without loss of input rna.
Primescript rt reagent kit perfect real time from takara. Both mrna and gdna were detected with the human tuba assay. Rr047a takara, shiga, japan which can digest the residual dna in the rna extractions to enhance the accuracy of qrtpcr. Reverse transcription quantitative pcr was used to detect the expression of mrna of hub genes by 7500 pcr system thermo fisher. Rna with a primescript rt reagent kit with gdna eraser perfect realtime. Singlestrand cdna was synthesized from 1 mg of total rna using the primescript rt reagent kit with gdna eraser takara biotechnology co. One step primescript rtpcr kit perfect real time 100.
Realtime polymerase chain reaction pcr was carried out. Tannic acid induction of a glutathione stransferase in. Primescript rt reagent kit with gdna eraser perfect. No amplification was observed from biorad iscript gdna clear cdna synthesis kit a, demonstrating complete removal of gdna. Because this realtime rtpcr kit is powered by primescript. Item primescript rt reagent kit perfect real time company takara bio. Primescript rt reagent kit with gdna eraser is a reversetranscription kit for realtime rtpcr rtqpcr that includes a genomic dna elimination reaction. Inhibition of cell proliferation and migration through. Overexpression of programmed death ligands in naturally. Primescript rt reagent kit with gdna eraser is a reversetranscription kit for realtime rt pcr rt qpcr that includes a genomic dna elimination reaction.
1040 1078 1349 1042 1395 1237 1475 881 1344 1016 1608 1532 1363 520 1629 1239 1000 1136 383 1293 301 624 893 894 1539 150 890 494 44 586 125 363 149 512 847 1242