Genes identified in the discovery phase were considered for validation by RT-qPCR, in addition to a reference gene—Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)—which was used to normalize the RNA input of each sample. Primers were designed as described in Supplementary Table 2 . The validation was performed using the Biomark HD (Fluidigm) and the 192.24 Dynamic Array integrated fluidic circuit (IFC) following manufacturer instructions. The IFC required the following 3 steps: reverse transcription, pre-amplification, and RT-qPCR (Supplementary Methods ).
192.24 dynamic array
Manufactured by Standard BioTools
Sourced in United States
The 192.24 Dynamic Array is a lab equipment product from Standard BioTools. It is designed to enable high-throughput analysis of up to 192 samples against 24 targets simultaneously. The core function of this product is to facilitate efficient and accurate gene expression analysis or targeted sequencing experiments.
Automatically generated - may contain errors
5 protocols using 192.24 dynamic array
1
Validation of Candidate Genes by RT-qPCR
2
High-throughput RT-qPCR Using BioMark HD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RT-qPCR was performed on the high-throughput BioMark HD system using a 192.24 Dynamic Array (Fluidigm, South San Francisco, CA, USA). All of the samples were pre-amplified for 17 cycles before the initial run, following the manufacturer’s protocol, and the final product was diluted fivefold. The samples and four negative control samples (ddH2O) were loaded in duplicates according to Fluidigm’s EvaGreen DNA-binding dye protocol onto the BioMark HD system. The following qPCR conditions were used: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 20 s, plus a melting curve analysis. The data were processed using the software Fluidigm Real-Time PCR analysis 4.0 (Fluidigm, South San Francisco, CA, USA). The quality threshold was set to the default value of 0.65. Furthermore, a linear baseline correction was performed, and the setting automatic detectors were used as the Ct threshold method.
3
Comprehensive Pathogen Detection in Porcine Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nucleic acids were extracted from faecal sock and oral fluid samples using the extraction robot QIAcube HT (QIAGEN, Hilden, Germany) and the Cador Pathogen 96 QIAcube HT kit (Indical Bioscience, Leipzig, Germany) using the manufacturer’s instructions. Positive and negative (nuclease-free water; Amresco, Cleveland, OH) controls were included in each extraction. The nucleic acids were stored at − 80℃ until further analysis. Prior to high-throughput real-time PCR analysis, reverse transcription and preamplification were performed as described previously [35 ]. Target specific primers and probes against B. pilosicoli, E. coli F4 and F18, L. intracellularis, PCV2, porcine parvovirus, rotavirus A [45 (link)], and rotavirus B, C and H (primers not yet published) were applied on the faecal sock samples. For oral fluid samples target specific primers against A. pleuropneumoniae, G. parasuis, M. hyopneumoniae, M. hyorhinis, PCV2 and 3, P. multocida, porcine cytomegalovirus, S. suis, influenza virus A were applied [45 (link)].
The pre-amplified cDNA and DNA was stored at − 20 °C until high-throughput real-time PCR amplification, using the BioMark HD (Fluidigm, South San Franscisco, USA) and 192.24 dynamic array (DA) integrated fluidic circuit (IFC) system (Fluidigm) as previously described [45 (link), 46 (link)].
The pre-amplified cDNA and DNA was stored at − 20 °C until high-throughput real-time PCR amplification, using the BioMark HD (Fluidigm, South San Franscisco, USA) and 192.24 dynamic array (DA) integrated fluidic circuit (IFC) system (Fluidigm) as previously described [45 (link), 46 (link)].
4
High-Throughput qPCR using Fluidigm IFC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HT-qPCR was performed using a 192.24 Dynamic Array integrated fluidic circuit (IFC; Fluidigm Corporation, San Francisco, CA, USA). The assay mix consisted of 3 μl 2× Assay Loading Reagent (Fluidigm Corp.) added to 3 μl primer mix (forward and reverse, 10 μM). A sample pre-mix was prepared by combining 3 μl 2× SsoFast™ EvaGreen® Supermix with low ROX (Biorad, Cressier, Switzerland) and 0.3 μl 192.24 Delta Gene Sample Reagent (Fluidigm Corp.). Finally, 2.7 μl of each sample were added to 3.3 μl sample pre-mix. The IFC was loaded according to the manufacturer’s instructions [33 ]. Briefly, 3 μl of each assay and 3 μl of each sample were distributed to the respective inlet, and the IFC was loaded using the Juno Load Mix 192.24 GE script. The loaded IFC was transferred to the Biomark instrument and run with the GE 192x24 PCR + Melt v2 program, as follows: hot start 95 °C for 1 min, followed by 30 cycles of denaturation at 96 °C for 5 s, and annealing and elongation at 60 °C for 20 s. A melting curve analysis was performed with a temperature increase of 1 °C per 3 s from 60 to 95 °C.
5
High-Throughput qPCR Bacterial Culture Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HT-qPCR was performed using a 192.24 Dynamic Array integrated fluidic circuit (IFC; Fluidigm Corporation, San Francisco, CA, United States). DNA samples from pure bacterial cultures were diluted to 3 ng/μl prior to qPCR measurement. The assay mix consisted of 3 μl 2× Assay Loading Reagent (Fluidigm Corp.) added to 3 μl primer mix (forward and reverse, 10 μM). A sample pre-mix was prepared by combining 3 μl 2× SsoFastTM EvaGreen® Supermix with low ROX (Biorad, Cressier, Switzerland) and 0.3 μl 192.24 Delta Gene Sample Reagent (Fluidigm Corp.). Finally, 2.7 μl of each sample were added to 3.3 μl sample pre-mix. The IFC was loaded according to the manufacturer’s instructions (Fluidigm, 2015 ). Briefly, 3 μl of each assay and 3 μl of each sample were distributed to the respective inlet, and the IFC was loaded using the Juno Load Mix 192.24 GE script. The loaded IFC was transferred to the Biomark instrument and run with the GE 192x24 PCR+Melt v2 program, as follows: hot start 95°C for 1 min, followed by 30 cycles of denaturation at 96°C for 5 s and annealing and elongation at 60°C for 20 s. A melting curve analysis was performed with a temperature increase of 1°C per 3 s from 60 to 95°C.
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!