T7 promoter primer, amino-cholesteryl (5′- and 3′-)-conjugated DNA, 3′-Cy5 labeled DNA, monomeric siRNA, and primers for qRT-PCR were all obtained from Bioneer Co. (Korea), as well as 5′-phosphorylated ssDNA templates for RCT reaction of various RNA nanoparticles including Dsi RNPs. The branched PEI (25 kDa) polymer was purchased from Sigma-Aldrich (UK). All oligonucleotide sequences are listed in Table S1 .
Qrt pcr
Manufactured by Bioneer
The QRT-PCR (Quantitative Real-Time Polymerase Chain Reaction) is a laboratory equipment used for the amplification and quantification of specific DNA or RNA sequences. It combines thermal cycling and fluorescent detection to enable real-time monitoring of the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
T7 promoter primer, by Bioneer (1 mentions)
Monomeric sirna, by Bioneer (1 mentions)
Power taq pcr mastermix, by BioTeke (1 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Solarbio (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Amikacin, by Merck Group (1 mentions)
Linezolid, by Merck Group (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mueller hinton broth (mhb), by BD (1 mentions)
Mueller hinton agar (mha), by BD (1 mentions)
Skim milk, by BD (1 mentions)
Vancomycin, by Merck Group (1 mentions)
Amoxicillin, by Merck Group (1 mentions)
Ceftazidime, by Merck Group (1 mentions)
E z n a bacterial rna kit, by Omega Bio-Tek (1 mentions)
3 protocols using qrt pcr
1
Preparation of RNA Nanoparticles
2
Validating Differential ncRNA Expression in Placenta
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A total of 40 placenta samples (20 NGT and 20 GDM) were selected for validation of differentially expressed ncRNAs by quantitative real‐time polymerase chain reaction (qRT–PCR).
Total RNA extraction and concentration measurement were the same as the previous method. Complementary deoxyribonucleic acid (cDNA) was synthesized by using M‐MLV Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Subsequently, qRT–PCR (BIONEER, Daejeon, Korea) was performed to measure the expression levels of miRNAs, lncRNAs and circRNAs according to the manufacturer’s instructions. qRT–PCR was performed in a 20‐μL reaction volume, including 10 μL 2×Power Taq PCR Master Mix (BioTeke, Beijing, China), 0.3 μL SYBR Green (Solarbio, Beijing, China), 2 μL cDNA, 0.5 μL PCR Forward Primer (10 μmol/L), 0.5 μL PCR Reverse Primer (10 μmol/L) and 6.7 μL nuclease‐free water. The protocol was initiated at 94°C for 5 min, followed by 94°C (10 s), 60°C (20 s) and 72°C (30 s) for a total of 40 cycles. U6 and β‐actin were used as internal control for miRNAs, lncRNAs and circRNAs, respectively. All reactions were performed in three independent wells. The relative expression levels of differentially expressed ncRNAs were determined using the 2−△△Ct method21 . The primer sequences a listed in Table S1 .
Total RNA extraction and concentration measurement were the same as the previous method. Complementary deoxyribonucleic acid (cDNA) was synthesized by using M‐MLV Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Subsequently, qRT–PCR (BIONEER, Daejeon, Korea) was performed to measure the expression levels of miRNAs, lncRNAs and circRNAs according to the manufacturer’s instructions. qRT–PCR was performed in a 20‐μL reaction volume, including 10 μL 2×Power Taq PCR Master Mix (BioTeke, Beijing, China), 0.3 μL SYBR Green (Solarbio, Beijing, China), 2 μL cDNA, 0.5 μL PCR Forward Primer (10 μmol/L), 0.5 μL PCR Reverse Primer (10 μmol/L) and 6.7 μL nuclease‐free water. The protocol was initiated at 94°C for 5 min, followed by 94°C (10 s), 60°C (20 s) and 72°C (30 s) for a total of 40 cycles. U6 and β‐actin were used as internal control for miRNAs, lncRNAs and circRNAs, respectively. All reactions were performed in three independent wells. The relative expression levels of differentially expressed ncRNAs were determined using the 2−△△Ct method
3
Isolation and Characterization of SGB from Sanguisorba officinalis
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SGB (Figure 6 ) was isolated from Sanguisorba officinalis L., and identified by spectral and physicochemical methods; the purity of HPLC analysis was more than 98% [20 (link)]. Skim milk, Mueller–Hinton agar (MHA), and Mueller–Hinton broth (MHB) were purchased from Difco Laboratories (Baltimore, MD, USA). Crystal violet, linezolid, gentamicin, vancomycin, amikacin, amoxicillin, ceftazidime, and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). The E.Z.N.A. Bacterial RNA Kit was obtained from Omega Bio-Tek (Norcross, GA, USA). The sequences of primers used in qRT-PCR was purchased from Bioneer (Daejeon, Korea).
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