The reverse transcription was achieved using QuantiMir kit (System Biosciences, Mountain View, CA, USA), according to the manufacturer’s instructions, starting from 1 μg of total RNA. Quant Studio 12K Flex Real-Time PCR (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to detect the expression of miR395 with the following reaction mixture: 5 μL of Power Syber Green PCR master mix (Life Technologies, Carlsbad, CA, USA), 0.1 μL of forward primer, 0.1 μL of reverse primer, 1.4 μL of nuclease-free water, and 1 μL of each sample. The PCR program was set up with an initial denaturation at 95 °C for 10 min followed by 50 cycles at 95 °C for 15 s and 60 °C for 1 min. The primer sequence used to detect miR395 was obtained from [20 (link),21 (link),22 (link),23 ,24 (link),25 (link),26 (link),27 (link),28 (link),29 (link),30 (link),31 (link),32 (link),33 (link),34 (link),35 (link),36 (link),37 ,38 (link)]. The threshold method was used to analyze miR395 expression and the ΔCt values, calculated based on Hajizadeh et al. [39 (link)], involved the use of 18S rRNA as an internal standard.
Quantstudio 12k flex real time pcr
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio™ 12K Flex Real-Time PCR System is a high-throughput instrument designed for quantitative real-time PCR analysis. The system supports up to 12 samples in a reaction plate and provides fast, accurate, and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Quantimir kit, by System Biosciences (2 mentions)
Miscript 2 rt kit, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Power syber green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Primer express v3, by Thermo Fisher Scientific (1 mentions)
Expression suite software v1, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Veriti thermal cycler, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green supermix, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions)
Primer express software, by Thermo Fisher Scientific (1 mentions)
9 protocols using quantstudio 12k flex real time pcr
1
Quantification of miR395 Expression
2
Leonardite Effects on Sugar Beet Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Eight sugar beet genes were used to test leonardite effects on plants. Primer design with Primer Express V3.0 (Thermo Fisher Scientific) was done starting from mRNA sequences downloaded from RefBeet_1.21 . Table 2 shows the complete list of genes, their functional category, and gene product. Quantitative RT Real-Time PCR amplification and detection were conducted on a Quant Studio 12K Flex Real-Time PCR (Thermo Fisher Scientific) using qPCRBIO SyGreen 1-step kit (Resnova-PCR Biosystem). The 10 μl of reaction mixture contained 5 μl of SYBR Green, 0.5 μl retrotranscriptase, 0.4 μl of forward and reverse primers, 0.7 μl of nuclease-free water, and 1 μl of RNA. The threshold cycle (Ct) values obtained were normalized against the average transcript abundance of three housekeeping genes (Tubulin, Bv2_037220_rayf; GAPDH, Bv5_107870_ygnn; Histone H3, Bv6_127000_pera) using the formula: 2–ΔCt in which ΔCt is obtained from the difference between the Ct of the target gene and the Ct of the control gene (Livak and Schmittgen, 2001 (link); Schmittgen and Livak, 2008 (link)).
3
Quantification of miRNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Using the miScript II RT kit (Qiagen), 1 µg of total RNA was reverse-transcribed in 20 μl reactions. cDNA from total bone was diluted x8 and cDNA from hOBs was diluted x16; in both cases, 2 µl were assayed in 10 µl PCR reactions in 384-well plates using MiScript Syber Green PCR kit according to the protocol. The sequence of the mature miRNAs selected, according to the mirBase web site, was used as a forward primer and the Universal primer as a reverse. U6 snRNA was used as the reference gene for normalization. Amplification was performed in a QuantStudio 12 K Flex Real-Time PCR (Applied Biosystems), and the Expression Suite software was used both for determination of relative quantification (RQ) (by 2−ΔΔCt method) and for melting curve analysis.
4
Quantifying miR-320a Expression in hOBs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the post-transfection miR-320a expression levels in hOBs, total RNA was extracted 48 hours after transfection using the miRNeasy mini kit (Qiagen) according to manufacturer instructions. Then, 1 μg of total RNA was reverse-transcribed in 20 μl reactions using the miScript II RT kit (Qiagen). cDNA was diluted 1/8 and 2 μl were assayed in 10 μl qPCR reactions in 384-well plates using MiScript SYBR Green PCR kit according to the protocol. The mature miR-320a sequence, according to the mirBase web site, was used as a forward primer (5’-AAAAGCTGGGTTGAGAGGGCGA-3’ ) and the Universal primer as a reverse. U6 snRNA was used as the reference gene for normalization. All qPCR reactions for each sample were performed in triplicate. Amplification was performed in a QuantStudio 12K Flex Real-Time PCR (Applied Biosystems), and the ExpressionSuite software v.1.0.3 (Life Technologies) was used both for determination of relative quantification (RQ) (by 2-ΔΔCt method) and for melting curve analysis.
5
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from tissues with TRIzol® (Invitrogen, Carlsbad, CA, USA), following the manufacturer's recommendations. The first strand of cDNA was generated from 2 μg of total RNA extracted, using a commercially available kit (High‐Capacity cDNA Reverse Transcription Kits, Invitrogen, Foster City, CA, USA). Polymerase chain reaction (PCR) amplification was performed in duplicates with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in the QuantStudio 12K Flex Real‐Time PCR (Applied Biosystems), adopting the primers listed in Table1 . Gene expression was normalized to the ribosomal protein L27 (Rpl27) reference gene. Data were calculated using the 2−ΔΔCT method and are presented as fold changes in gene expression relative to control samples.
6
Transcriptional Profiling by Real-Time PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted with TRIzol® reagent (Invitrogen, Carlsbad, CA), following the manufacturer's recommendations. The concentration of total RNA was assessed with the Biotek® SynergyH1 spectrophotometer (Fisher Scientific, Biotek, Winooski, USA). The cDNA was obtained in a thermocycler (Veriti® Thermal Cycler; Applied Biosystems, Foster City, CA) using a high‐capacity cDNA reverse transcription kits (Invitrogen No. 4375575). Transcript levels of genes were evaluated by real‐time PCR with SYBR Green (Invitrogen) in Quant Studio™ 12K Flex real‐time PCR (Applied Biosystems Carlsbad, CA, USA). Primers employed for the amplification are described in Table1 . Gene expression was normalised against the housekeeping gene β2‐microglobulin (B2m). Relative gene expression was calculated by the comparative 2‐ΔΔCT method, described by Livak and Schmittgen.40
7
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from the tissues and cells, with Trizol® (Invitrogen, CA, USA), following the manufacturer's recommendations. The first strand of cDNA was generated from 2 µg of total RNA, employing a commercial kit (High Capacity cDNA Reverse Transcription Kit, Invitrogen). Polymerase chain reaction (PCR) amplification was performed in duplicates, with SYBR Green PCR Master Mix (Applied Biosystems, CA, USA) in the QuantStudio™ 12 K Flex Real Time PCR (Applied Biosystems, CA, USA), employing the primers listed in Table1 . Gene expression was normalized to the Rpl19 and Rpl27 reference genes. Data were calculated with the 2‐ΔΔCT method and are presented as the fold change in gene expression relative to the control sample.
8
RNA Isolation and qPCR Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RNA isolation, the Qiagen RNeasy Plus Mini kit was used according to the manufacturer’s instructions.After isolation of RNA, samples were diluted in 20–40ul RNase free water, depending on target RNA concentrations desired. Total RNA quantification and RNA purity were determined based on the ratio A260nm/A280 nm using Thermo Fisher Scientific NANO drop 2000. A total of 500 to 1000ng of cDNA was synthesized using the iScript cDNA synthesis kit (BioRad, #1708891). 10ng cDNA was used for quantitative PCR in technical triplicates in a 384 well plate using SYBR Green Supermix (Applied Biosystems). PCR was run in Applied Biosystems QuantStudio™ 12K Flex Real-Time PCR at the Advanced Genomics Core at the University of Michigan. Primer sequences are summarized in Supplemental Table 4 . Gene expression level was determined by relating to the housekeeper gene beta-Actin or RPLP0 using the ∆∆ct method setting the mock condition to one.
9
Quantitative Transcriptional Profiling of Host
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RT-qPCR assays was performed on lung and liver samples collected on dpi 0, 4, 7, and 10. The RNA was reverse-transcribed with the High-Capacity cDNA Archive Kit (Life Technologies, Courtaboeuf, France). The resulting cDNA was amplified using SYBR Green-based real-time PCR and QuantStudio™ 12K Flex Real-Time PCR systems (Applied Biosystems™, Villebon-sur-Yvette, France), according to the manufacturer’s instructions. The expression was normalized against the ACTG1 housekeeping gene encoding γ-actin. Specific primers were designed using Primer Express software (Applied Biosystems) and ordered from Eurofins Scientifics (Ebersberg, Germany) (Table 1 ). Relative mRNA levels (2−∆∆Ct) were determined by comparing the respective PCR cycle thresholds (Ct) for the gene of interest and the housekeeping gene (∆Ct) and those for the infected vs. mock groups (∆∆Ct). Viral RNA loads were normalized against γ-actin expression levels (∆Ct).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!