Total RNA was extracted using either the QIAsymphony® RNA robotic system (Qiagen) (challenge material; 5 mg hind gut and gill, 20 mg pectoral fin and skin/mucus swabs) or the RNeasy tissue kit (Qiagen) (ISA outbreak samples; 5 mg) according to manufacturer’s protocols. For hind gut and all ISA outbreak samples, cDNA was synthesized using the TaqMan® Reverse Transcription Reagents kit (Life Technologies, UK) with oligo-d(T)16 as described previously [21 (link)] and real-time PCR (qPCR) analysis was performed on a Lightcycler LC96 (Roche) as described in [6 (link)] using assays targeting ELF and ISAV segment 8 [7 (link)]. For skin swabs and pectoral fin, one-step real-time RT-qPCR (Quantitect Probe RT-PCR kit, Qiagen) was performed as outlined in McBeath et al. [7 (link)]. Transcription of immune genes type I and II IFN, Mx and γIP were analysed on a Lightcycler LC96 (Roche) for all tissues and statistical analysis was performed as previously described [7 (link)]. RNA species specific RT and qPCR to specifically examine replication (cRNA) and transcription (mRNA) was performed according to McBeath et al. [7 (link), 22 (link)].
Lightcycler lc96
Manufactured by Roche
Sourced in Australia
The LightCycler LC96 is a real-time PCR system designed for nucleic acid amplification and detection. It features a high-performance optical system, automated sample handling, and an intuitive user interface. The LightCycler LC96 is capable of performing quantitative and qualitative real-time PCR analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rnalater, by Qiagen (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Faststart essential dna green master, by Roche (2 mentions)
Sybr green 1 dye, by Roche (2 mentions)
High pure rna isolation kit, by Roche (2 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (2 mentions)
Calcein, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr instrument, by Bio-Rad (1 mentions)
Mncl2, by Merck Group (1 mentions)
Sybr green pcr kit, by Qiagen (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rneasy tissue kit, by Qiagen (1 mentions)
Qiasymphony rna robotic system, by Qiagen (1 mentions)
Taqman reverse transcription reagents kit, by Thermo Fisher Scientific (1 mentions)
Quantitect probe rt pcr kit, by Qiagen (1 mentions)
Quantifast sybr green rt pcr kit, by Qiagen (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Rneasy column, by Qiagen (1 mentions)
Lightcycler 96 sw 1, by Roche (1 mentions)
10 protocols using lightcycler lc96
1
Comprehensive RNA extraction and analysis for disease detection
2
Optimized LAMP Assay for HPV Detection
3
Islet and Placenta RNA Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated female islets from non-pregnant and pregnant (day 16) mice were immediately snap frozen in liquid nitrogen following purification from the exocrine pancreas for subsequent RNA extraction using RNeasy Mini Kit (Qiagen) and High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) for cDNA synthesis, as described previously (Drynda et al. 2018 (link)). Placenta samples were also collected after termination at day 18 of pregnancy and snap frozen. RNA extraction and cDNA conversion were conducted as described earlier. Islet CRH receptor and placental CRH ligand mRNA expression were subsequently quantified by quantitative RT PCR (qRT-PCR) using SYBR Green PCR Kit (QuantiTect, Qiagen) and a LC96 Light Cycler (Roche Diagnostics). QuantiTect primer assays were used for expression analysis of genes of interest using glyceraldehyde 3-phosphate dehydrogenase (Gapdh) as the housekeeping gene (Mouse Crh-QT01055789, Ucn1-QT00326879, Ucn2-QT01556534, Ucn3-QT00302267, Crhr1-QT00106232, Crhr2-QT00151543, Gapdh-QT01658692, Qiagen).
4
Quantitative RT-PCR of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cells. cDNA was synthesized using standard techniques. PCR was performed using a Roche LightCycler LC96. Ct values were normalized to housekeeping gene S16, and fold changes of gene expression relative to vehicle control non-asthmatic fibroblasts were calculated by the ΔΔCt method. Primers used were from IDT. The primer sequences are listed in Supplementary Table S2 .
5
RT-qPCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples were preserved in RNAlater (Qiagen) and stored at −80 °C. The rings surrounding ear pinna holes were excised with a 3-mm biopsy punch. RNA was extracted using an RNeasy Kit (Qiagen). cDNA synthesis was performed in a reaction mix containing 200 ng of RNA, 100 pmoles of oligo dT20, 4 μL of 5× reaction buffer (250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2, 50 mM DTT), and 200 units of Maxima Reverse Transcriptase (ThermoScientific Bio, Cat. No. EP0742) in a final volume of 20 μL. Real-time PCR was carried out in a final volume of 10 μL containing 5 μL of FastStart Essential DNA Green Master (Roche, Cat. No. 06402712001), 1 μL of cDNA, and 0.25 μL each of forward and reverse primers (10 μM) on a LightCycler LC96 (Roche). The transcript levels were calculated using the 2-∆∆Ct method relative to Actb and Tbp (mouse templates) and ACTB, GAPDH and TBP (human templates). The primer sequences are listed in Supplemental Information, Table S1. PCR was performed in triplicate.
6
RT-qPCR Analysis of Apoptosis-related Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA extraction was performed using High Pure RNA Isolation Kit (Roche Applied Science, Castle Hill, Australia). 1 μg RNA was used in a 20 μl reverse transcription reaction using Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Castle Hill, Australia). RT-qPCR was carried out in duplicate with cDNA using SYBR Green I dye (Roche Applied Science, Castle Hill, Australia) and performed using LightCycler LC96 (Roche Applied Science, Penzberg, Germany). All primers were purchased from Sigma-Aldrich (Castle Hill, Australia). Relative quantification using E-method established by Roche Applied Science was performed with β-Actin mRNA as reference sequence and untreated samples as study calibrator. cDNA samples were amplified using the following primer pairs with amplification efficiency (E): β-Actin: 5'-ACTCTTCCAGCCTTCCTTC-3' (forward) and 5'-GATGTCCACGTCACACTTC-3'(reverse), E=1.72; Bcl-2:
5'-ATGGGATCGTTGCCTTATGC-3'(forward) and
5'-CAGTCTACTTCCTCTGTGATGTTGT-3' (reverse), E=1.89; CDK9:
5'-AAAACGAGAAGGAGGGGTTCC-3' (forward) and
5'-CCTTGCAGCGGTTATAGGGG-3' (reverse), E=1.92; Mcl-1:
5'-AACAAAGAGGCTGGGATGGG-3' and 5'-TGCCAAACCAGCTCCTACTC-3' (reverse), E=1.80; Mnk1: 5'-AAGGCCATTGAGACACTTCG-3' (forward) and 5'-CCCAAATGAAATAAAGCTCCTG-3' (reverse), E = 1.74.
5'-ATGGGATCGTTGCCTTATGC-3'(forward) and
5'-CAGTCTACTTCCTCTGTGATGTTGT-3' (reverse), E=1.89; CDK9:
5'-AAAACGAGAAGGAGGGGTTCC-3' (forward) and
5'-CCTTGCAGCGGTTATAGGGG-3' (reverse), E=1.92; Mcl-1:
5'-AACAAAGAGGCTGGGATGGG-3' and 5'-TGCCAAACCAGCTCCTACTC-3' (reverse), E=1.80; Mnk1: 5'-AAGGCCATTGAGACACTTCG-3' (forward) and 5'-CCCAAATGAAATAAAGCTCCTG-3' (reverse), E = 1.74.
7
Quantitative Analysis of WTAP and BTV mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BUcEC were directly lysed in 0.35 mL of RLT buffer (QIAGEN) containing 1% 2-Mercaptoethanol. Total RNA was extracted using RNeasy columns (QIAGEN) according to the manufacturer’s instructions. RT-qPCR was performed using Quantifast SYBR Green RT-PCR Kit (Qiagen) with 50 ng of total RNA and experimental or control primers. The sequences of experimental primers targeting WTAP and the BTV segment 1 were 5′-CTACTCAGATCCAGTACCTCAAGCAA-3′ and 5′-CATTTTGGGCTTGTTCCAGTTT-3′, and 5′-GTTCCGCGCTAAAAACGAGA-3′ and 5′-CCCTGGTGGAATGGTGAATC-3′, respectively. Target transcripts were normalized to the control housekeeping gene, GAPDH, and the sequences used were 5′-GGTCGGAGTGAACGGATTCG-3′ and 5′-ACTCCACCACATACTCAGCA-3′. Reactions were performed using the LightCycler LC96 (Roche) and expression levels were analyzed using LightCycler 96 SW1.1 (Roche). The expression of WTAP and the BTV segment 1 were first normalized to the expression of GAPDH, then to the target gene expression of siCTR samples to calculate 2−Δ(ΔCT) relative expression values, as specified in the figure legend.
8
Quantifying Gene Expression in Skin Wounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prior to tissue collection, the animals were euthanized in a CO2 chamber. The tissue surrounding dorsal skin wounds was excised with a 4 mm biopsy punch. Tissue samples were preserved in RNAlater (Qiagen) and stored at −80°C. RNA was extracted using an RNeasy Kit (Qiagen). cDNA synthesis was performed in a reaction mix containing 200 ng of RNA, 1 μl of oligo dT20 (100 μM), 1 μl of dNTP mix (10 mM each), 4 μl of 5× reaction buffer (250 mM Tris‐HCl, 375 mM KCl, 15 mM MgCl2, 50 mM DTT), and 200 units of Maxima Reverse Transcriptase (Thermo Scientific Bio, Cat. No. EP0742) in a final volume of 20 μl. Real‐time PCR was carried out in a final volume of 10 μl containing 5 μl of FastStart Essential DNA Green Master (Roche, Cat. No. 06402712001), 2 μl of cDNA, and 0.25 μl each of forward and reverse primers (10 μM) on a LightCycler LC96 (Roche).PCR was performed in triplicate. The transcript levels were calculated using the 2−∆∆Ct method relative to Actb and Tbp transcript levels. The primer sequences are listed in Table 2 . The qPCR thermal profile was as follows: preliminary denaturation at 95°C for 10 min, followed by 40 cycles consisting of denaturation at 95°C for 10 s, annealing at 70°C for 10 s with touch‐down every 1.0°C per cycle, and primer extension at 72°C for 10 s, followed by a final extension at 72°C for 10 min.
9
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from whole worm lysate from a just starved plate using Sigma TRI reagent. cDNA was synthesized using Superscript IV Reverse Transcriptase (ThermoFisher Scientific) using random hexamers. qPCR was done with Kapa SYBR FAST universal qPCR mastermix on Roche LightCycler- LC96. For purposes of quantitation, the samples were normalized to wild type. At least three independent biological repeats were carried out with at least two technical repeats. No genomic DNA contamination was observed when using actin primers. Quantitation was carried out as described in61 (link).
10
Quantification of Mnk1 and Mnk2 Transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from the cells using a High Pure RNA Isolation Kit (Roche Applied Science, Castle Hill, NSW, Australia) and 1 μg of RNA was used in a 20 μL reverse transcription reaction (Transcriptor First Strand cDNA Synthesis Kit, Roche Applied Science, Castle Hill, NSW, Australia). RT-qPCR was performed in duplicate using SYBR Green I dye (Roche Applied Science, Castle Hill, NSW, Australia) with the LightCycler LC96 (Roche Applied Science, Penzberg, Germany). All primers were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Relative quantification using E-method established by Roche Applied Science was performed with β-actin mRNA as reference sequence and untreated samples as study calibrator. The cDNA samples were amplified using the following primer pairs with amplification efficiency (E): β-actin: 5′-ACTCTTCCAGCCTTCCTTC-3′(forward) and 5′-GATGTCCACGTCACACTTC-3′(reverse), E = 1.70; Mnk1: 5′-AAGGCCATTGAGACACTTCG-3′(forward) and 5′-CCCAAATGAAATAAAGCTCCTG-3′(reverse), E = 1.74; Mnk2: 5′-TCCTGCAGAGGTGGGACAGT-3′(forward) and 5′-ACGGTTCTGACCAGTCCTCC-3′ (reverse), E = 1.75.
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!