Total RNA was extracted from NSCs using TRIzol (#15596018, Thermo Fisher Scientific). A PrimScript RT Reagent Kit with gDNA Eraser (#RR047A, Takara) was used for cDNA synthesis. All primers were shown in Supplementary Table S1 . RT-PCR was conducted using TB Green Premix Ex Taq II (#RR820, Takara) on LightCycler 480 II system (Roche). Data were collected and analyzed by the LightCycler 480 software (1.5.0).
Primscript rt reagent kit with gdna eraser
Manufactured by Takara Bio
Sourced in Japan
The PrimScript RT reagent Kit with gDNA Eraser is a laboratory product designed for the reverse transcription of RNA. It includes a gDNA Eraser component to eliminate genomic DNA contamination prior to cDNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Tb green premix ex taq 2, by Takara Bio (2 mentions)
Lightcycler 480 2 system, by Roche (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Bcip nbt, by Merck Group (1 mentions)
Alkaline phosphatase conjugated goat anti mouse igg, by Vector Laboratories (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti p akt 4060s, by Cell Signaling Technology (1 mentions)
Anti n cadherin ab76011, by Abcam (1 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taq kit, by Takara Bio (1 mentions)
Sybr premix ex taqtm 2, by Takara Bio (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht, by Thermo Fisher Scientific (1 mentions)
9 protocols using primscript rt reagent kit with gdna eraser
1
Quantitative RT-PCR Analysis of NSCs
2
Salamander Muscle Transcriptome and Proteome Analysis
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Muscle tissues at the injection site were taken from Chinese giant salamanders at seven days after vaccination, frozen in liquid nitrogen, and stored at −80 °C until used. Total RNA was extracted using TRIzol reagent (Invitrogen) based on the manufacturer’s instructions. Two micrograms of RNA were then reverse transcribed into cDNA using the PrimScript™ RT reagent Kit with gDNA Eraser (Takara, Tokyo, Japan). PCR was carried out with specific primers 2L-F2/R2 and 58L-F2/R2 (see Table S1 ). The β-actin gene was used as an internal control.
Total protein lysates were prepared through the homogenization of frozen muscle tissues in 50 mM Tris-HCl buffer (pH 7.5) with a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Lysates were centrifuged at 12,000× g for 20 min and the pellets were discarded. Samples were blotted onto the polyvinylidene difluoride (PVDF) membrane for immunodot blot. Mouse anti-ADRV 2L and anti-ADRV 58L sera were used as primary antibodies, and alkaline phosphatase-conjugated goat anti-mouse IgG (Vector Laboratories) was used as a secondary antibody. The blots were developed with NBT/BCIP (Sigma). ADRV suspension from infected EPC cell cultures was used for a positive control.
Total protein lysates were prepared through the homogenization of frozen muscle tissues in 50 mM Tris-HCl buffer (pH 7.5) with a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Lysates were centrifuged at 12,000× g for 20 min and the pellets were discarded. Samples were blotted onto the polyvinylidene difluoride (PVDF) membrane for immunodot blot. Mouse anti-ADRV 2L and anti-ADRV 58L sera were used as primary antibodies, and alkaline phosphatase-conjugated goat anti-mouse IgG (Vector Laboratories) was used as a secondary antibody. The blots were developed with NBT/BCIP (Sigma). ADRV suspension from infected EPC cell cultures was used for a positive control.
3
Western Blot Assay for S100A11
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The antibodies that we used for the Western Blot assay are as follows: Primary antibody: anti-S100A11 (10237-1-AP) was purchased from Proteintech (USA), anti-GAPDH (P04406), anti-E-cadherin(#14472), anti-ERK (#4695T), anti-p-ERK (#4370T), anti-AKT (#5691S) and anti-p-AKT (#4060S) were from Cell Signaling Technology (Shanghai, China), anti-N-cadherin (ab76011) was from Abcam company (MA, United States); Secondary antibodies: anti-mouse IgG, HRP-linked antibody(#7076) and mouse anti-rabbit IgG mAb (HRP conjugate) were also purchased from Cell Signaling Technology (Shanghai, China). PrimScript RT reagent Kit with gDNA Eraser (RR047A) was from Takara (Japan), and SYBR Green PCR Master Mix was from Thermo Fisher Scientific (USA).
4
Quantitative PCR for Gene Expression
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Total RNA was extracted from cultured cells using the Gene JET RNA Purification Kit (Thermo Fisher Scientific, #K0731) according to the manufacturer's instructions. A total of 12.5 ng cDNA synthesized from 1 μg of total RNA using the PrimScript RT reagent Kit with gDNA Eraser (TaKaRa, #RR047A) was subjected to qPCR using a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). The 2−ΔΔCT method was used to calculate the relative gene expression levels normalized against an average of three housekeeping genes (GAPDH, β-actin, and 18s rRNA). Primer sequences are listed in Supplementary Table S5.
5
Quantitative Real-Time PCR Transcriptome Analysis
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RNA samples from seven time points after cross‐cutting collected from Ll and Lr during the early VP process were applied for a qRT‐PCR. A PrimScript RT reagent Kit with gDNA Eraser (Takara, Otsu, Japan) was used for cDNA synthesis. The products were diluted 20‐fold with sterile water and used as templates, with primers designed using beacon designer , version 7.9 (Table S7 ) (Li et al., 2022 (link)). qRT‐PCRs were performed with a TB Green Premix Ex Taq Kit (RR420A; TaKaRa, Tokyo, Japan) in a Connect™ Optics Module (Bio‐Rad, Hercules, CA, USA). The expression levels of the tested transcripts were calculated by the 2−∆∆Ct method (Livak & Schmittgen, 2001 (link)). The values are expressed as the mean of three biological replicates.
6
Cardiac Total RNA Extraction and qPCR
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Total RNA was extracted from heart tissues using TRIzol™ reagent (Invitrogen, 15596-026). Equal quantities of RNA (1 μg) were converted into cDNA with a PrimScript™ RT reagent kit with gDNA Eraser (Takara, RR047A). Quantitative TaqMan PCR was performed with SYBR Premix Ex TaqTM II (Takara, RR082A) tested by an Applied Biosystems 7500 Real Time PCR System. All data were normalized to GAPDH.
7
Quantitative gene expression analysis in maize and Arabidopsis
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Total RNA of tissues of maize and Arabidopsis were extracted using TRIZOL reagent (Invitrogen, USA) and PrimScript RT reagent Kit with gDNA eraser was used for cDNA reverse transcription (TAKARA, Japan). Approximately 1 µg of total RNA was used for DNaseI treatment and cDNA synthesis, and quantitative real-time PCR (qRT-PCR) analysis was performed with an ABI 7500 real-time PCR system (Applied Biosystems, USA) using SYBR Premix Ex Taq II (TaKaRa, Japan) and gene-specific primers. AtUBQ10 was used as an internal control for Arabidopsis, and ZmFPGS was used for normalization of maize RNAs. Each qPCR assay was replicated for three biological replicates, and each biological replicate was examined in triplicate (Wang et al., 2019 (link)). The sequences of the primers are shown in Supplementary Table S1
8
RNA Isolation and qRT-PCR Analysis
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Total RNA was isolated using a modified Trizol extraction protocol49 (link). First-strand cDNA was synthesized using PrimScript™ RT reagent Kit with gDNA Eraser (Takara). Quantitative real-time RT-PCR was performed on ABI Prism 7900HT (Applied Biosystems) with the SYBR Green system. The gene specific primers used in qRT-PCR analysis were listed in Supplementary Table S6 . The Actin gene was used as internal control.
9
Quantitative RT-PCR of CD45+ Cells
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Total RNA was extracted from CD45+ cells sorted from spinal cords using TRIzol (Thermo Fisher) with standard or ActD-based dissociation methods. cDNA synthesis was performed using the PrimScript RT Reagent Kit with gDNA Eraser (Takara) according to the manufacturer’s protocol. Quantitative RT–PCR was performed using 100 ng cDNA and TB Green Premix Ex Taq II (Takara) on LightCycler 480 II system (Roche) with a cycling program of 95 °C for 30 s, followed by 40 cycles of 5 s at 95 °C, and 1 min at 60 °C. A melting curve of the amplified products was obtained using the above program. Data was collected and analyzed using the LightCycler 480 Software (1.5.0).
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