One-thousand five-hundred ng of RNA [a mix of the 10- and 30-hr time points from the third RNA-Seq time course published in the work by Hurley et al. (2014) chosen to encompass maximal expression of genes at two different clock phases] was used to prepare cDNA using the SuperScript III First-Strand synthesis kit (Invitrogen, Waltham, MA). This was followed by RT-PCR using the Fast SYBR green master mix kit in an ABI 7500 real-time cycler (Applied Biosystems, Waltham, MA). The primer combinations used are listed in Table S1 and the final concentration of primers in the reaction mix was 0.5 μM. The following cycling parameters were used: step 1: 95° for 5 min and step 2: 95° for 10 sec and 60° for 30 sec for 40 cycles. Ct values were calculated using software provided by the instrument manufacturer. The relative mRNA levels for each time point were calculated using at least two out of three biological replicates in the case where one of the replicates differed from either of the other two by more than three-fold. Only a single biological replicate and primer concentration was tested.
Abi 7500 real time cycler
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 7500 real-time cycler is a laboratory instrument designed for quantitative real-time PCR (qRT-PCR) analysis. It is capable of performing high-throughput nucleic acid detection and quantification across a wide range of sample types and applications.
Automatically generated - may contain errors
Lab products found in correlation
Fast sybr green master mix kit, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Quantitect sybr green rt pcr master mix, by Qiagen (1 mentions)
Universal cdna synthesis kit, by Qiagen (1 mentions)
Hid real time pcr analysis software v1, by Thermo Fisher Scientific (1 mentions)
Genelute mammalian total rna miniprep kit, by Merck Group (1 mentions)
Iscript one step rt pcr kit with sybr green, by Bio-Rad (1 mentions)
6 protocols using abi 7500 real time cycler
1
Quantifying Circadian Gene Expression
2
Quantifying Neurotrophin Signaling in Epithelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from TiO2‐NP pre‐exposed bronchial epithelial cells infected with rrRSV and nonexposed control cells using the RNeasy kit (Qiagen, Valencia, CA). This RNA was used for RT‐PCR analysis using the SYBR green one‐step RT‐PCR master mix (Qiagen) in an ABI 7500 real‐time cycler (Applied Biosystems). Validated NGF, TrkA and p75NTR primers were purchased from SABiosciences (Rockville, MD). Relative expression was calculated for each sample using the ΔΔCt method after normalization against the endogenous controls hypoxanthine phosphoribosyltransferase‐1, peptidylprolyl isomerase A (cyclophilin A), and beta‐2‐microglobulin (HPRT1, PPIA, and B2M, respectively; RealTimePrimers, Elkins Park, PA).
3
Quantification of Neurotrophic Factors in Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung tissue expression of genes encoding for key neurotrophic factors and their cognate receptors was conducted as described previously (26 (link)). Briefly, frozen lung specimens were homogenized in RLT buffer (Qiagen, Valencia, CA). Total RNA was isolated using an RNeasy kit following manufacturer's recommendations, and 100 ng was used as the template for quantitative real-time PCR analysis using Quantitect SYBR green RT-PCR master mix (Qiagen). Samples were run with the ABI 7500 real-time cycler (Applied Biosystems, Foster City, CA). Primers for NGF and BDNF; TrKA and TrKB; and the nonspecific pan-neurotrophin receptor p75NTR were purchased from SuperArray (Rockville, MD), and hypoxanthine phosphoribosyltransferase-1 (Real Time Primers, Elkins Park, PA) was used as the housekeeping gene for transcript normalization. Relative changes in gene expression were calculated with the 2−ΔΔCT method.
4
Quantification of miRNA Profiles in uEVs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four nanograms of total RNAs were retrotranscribed according to Universal cDNA Synthesis Kit (#203301 Exiqon, Vedbaek, DK) protocol. cDNAs were diluted at 1:40 and combined with SYBR Green master mix following the miRCURY LNA Universal microRNA PCR (#203403 Exiqon, Vedbaek, DK) instructions. qPCR was performed in an ABI7500 Real-Time cycler (Applied Biosystems). Primers used were designed to detect: miR-200c-3p, miR-103a-3p, miR-let7b-5p, miR-99a-5p and miR-10b-5p. Unisp6 was added as an exogenous control, since they have been found in uEVs [27 (link)].
5
Quantifying ZIKV RNA by RT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ZIKV RNA copies were quantified using one-step RT-PCR. Viral RNA was extracted from culture pellet using GenElute Mammalian Total RNA Miniprep kit (Sigma, St. Louis, MO) according to the manufacturer's protocol. Two sets of primer pairs were designed to target the 5′UTR region of the virus genome (Forward primer, ZIKV_F, 5′-TTGGTCATGATACTGCTGATTGC-3′ and reverse primer, ZIKV_R, 5′-CCTTCCACAAAGTCCCTATTGC-3′) and (Forward primer, Zika E_F, 5′AAGTTTGCATGCTCCAAGAAAAT-3′ and reverse primer, Zika E_R,-5′CAGCATTATCCGGTACTCCAGAT-3′). The reactions of qRT-PCR were carried out using iScript One-step RT-PCR kit with SYBR Green (Bio-Rad, Hercules, CA) and quantification was performed using ABI 7500 real-time cycler (Applied Biosystems, Foster City, CA). The thermal cycling profile of this assay consisted of a 10 min cDNA synthesis step at 50 °C, 5 min of iScript reverse transcriptase inactivation at 95 °C, followed by 35 cycles of PCR at 95 °C for 10 s and a step of a single fluorescence emission data collection at 59 °C for 30 s. Each experiment was repeated three times with low MOI and two times with high MOI of ZIKV. Results were analyzed using HID Real-Time PCR Analysis Software v1.2 (Thermo Scientific, Waltham, MA). Relative degree of ZIKV replications in different cell lines were calculated using cycle threshold values (Ct).
6
Quantitative FeLV Proviral Load Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The FeLV qPCR test was performed as previously described [49 (link)] using a unique region within the U3 of the FeLV LTR with a reported detection limit of 1000 copies per reaction. Specificity of the assay was 100% by obtaining negative results for PBMC DNA from 80 pathogen-free, FeLV-negative cats. Assay sensitivity was also 100% by confirmation of infection in 30 FeLV p27-positive cats. Cats that were experimentally infected and that became persistently antigenaemic as determined by p27 ELISA were considered positive.
The primers and probe FeLV-U3-exo-f 5′-AAC AGC AGA AGT TTC AAG GCC-3′ (forward), FeLV-U3-exo-r 5′-TTA TAG CAG AAA GCG CGC G-3′ (reverse) and FeLV-U3-probe 5′-FAM-CCA GCA GTC TCC AGG CTC CCC A-TAMRA-3′ were used to detect a 131-bp LTR sequence. Standard curves were performed with a plasmid containing the target region in a serial dilution range of 103–109 copies. The cycling conditions consisted of an initial step of GoTaq (Promega, Madison, WI, USA) activation at 95 °C for 2 min followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C using an ABI 7500 real-time cycler (Applied Biosystems, Foster City, CA, USA). The number of pVL copies/cell was calculated similarly to the FFV pVL, except the pVL was divided by two to compensate for the presence of two LTR copies per integrated provirus.
The primers and probe FeLV-U3-exo-f 5′-AAC AGC AGA AGT TTC AAG GCC-3′ (forward), FeLV-U3-exo-r 5′-TTA TAG CAG AAA GCG CGC G-3′ (reverse) and FeLV-U3-probe 5′-FAM-CCA GCA GTC TCC AGG CTC CCC A-TAMRA-3′ were used to detect a 131-bp LTR sequence. Standard curves were performed with a plasmid containing the target region in a serial dilution range of 103–109 copies. The cycling conditions consisted of an initial step of GoTaq (Promega, Madison, WI, USA) activation at 95 °C for 2 min followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C using an ABI 7500 real-time cycler (Applied Biosystems, Foster City, CA, USA). The number of pVL copies/cell was calculated similarly to the FFV pVL, except the pVL was divided by two to compensate for the presence of two LTR copies per integrated provirus.
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!