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RNA Isolation and qPCR.
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RNA was isolated from skeletal muscle samples (~10 mg) as demonstrated previously (17). Muscle tissue was homogenized in TriReagent following specific manufacturer instructions for aqueous phase separation and precipitation and washing of the RNA pellet (RT 111; Molecular Research Center, Cincinnati, OH). RNA concentration was quantified and assessed for RNA purity using the 260/280 nm ratio (1.88 ± 0.04). Approximately 1 μg of total RNA was synthesized using a commercially available kit (iScript, Bio-Rad, Hercules, CA). Real-time PCR was carried out with a CFX Connect real-time PCR cycler (Bio-Rad) under similar protocol conditions as reported previously (17) using either TaqMan fluorescence predesigned or SYBR green custom designed primers. Each reaction tube consisted of a SYBR Green or TaqMan master mix along with ultrafiltered water and specific forward and reverse primers for a total reaction volume of either 25 or 20 μl, respectively. For SYBR green, we used the following PCR temperature conditions: 1 cycle for 3 min at 95°C followed by 40 cycles at 95°C for 20 s and at 55°C (or 60°C) for 30 s. TaqMan PCR assays (Thermo Fisher Scientific, Waltham, MA) were performed under the recommended PCR temperature guidelines: 1 cycle for 10 min at 95°C followed by 40 cycles at 95°C for 15 s and at 60°C for 60 s. The following TaqMan primers were used: TLR2 (Hs01872448_s1), RAGE (Hs00179504_m1), TAK1 (Hs00177373_m1), IL6 (Hs00985639_m1), and HMGB1 (Hs1923466_g1). MYD88, MCP1, NFKB1 and β2M primers have been designed and published previously (18, 37). The remaining SYBR green primers were designed for this study using standard optimization guidelines (58): TLR4 (NM_138554) (Fwd: 5′-TTCTTCTAACTTCCTCTCCTGTGA-3′, Rev: 5′-AGCGGCAACCTTAGCATTC-3′), TNF associated factor 6, E3 ubiquitin protein ligase (TRAF6; NM_145803) (Fwd: 5′-TTTGCTCTTATGGATTGTCCCC-3′ Rev: 5′-CATTGATGCAGCACAGTTGTC-3′), serine palmitoyltransferase long chain base subunit 1 (SPT1; NM_006415) (Fwd: 5′-TTCCAGTCTTCGTTGTGT-3′, Rev: 5′-GGCTAAGGATGCTCAGAT-3′), serine palmitoyltransferase long chain base subunit 1 (SPT2; NM_004863) (Fwd: 5′-AAGAATCCAGCCATCGTA-3′, Rev: 5′-ATGACTACAGAAGCAAGAATC-3′), ceramide synthase 1 (CERS1; NM_021267) (Fwd: 5′-CCATCTCCGTGCTCTTCTT-3′, Rev: 5′-CACTCGTCCACCACCATG-3′), ceramide synthase 2 (CERS2; NM_181746) (Fwd: 5′-TGGCAATAAGTGTCAGAC-3′, Rev: 5′-GCAAGGAAGGCATAAGAA-3′), and sphingosine kinase 1 (SPHK1; NM_021972) (Fwd: 5′-TATGAATGCCCCTACTTG-3′, Rev: 5′-TCGCTAACCATCAATTCC-3′). All designed primers were carefully optimized for efficiency (between 90 and 110%) and verified by melt analysis and product size using a DNA agarose gel. Cycle threshold values were normalized to beta 2-microglobulin (β2M) for comparisons of Pre vs. Post exercise training in HipFx. However, due to difficulties identifying a stable housekeeping gene (β2M, GAPDH, and HMBS) or calculating geometric means between HipFx and CON and in the interests of preserving limited cDNA, we used chemokine receptor type 2 (CCR2; Hs00704702_s1) as a normalizing gene because it remained stable across the exercise intervention and between groups. Fold change values were calculated using the 2−ΔΔCt method. In the case of comparing Pre vs. Post HipFx gene expression, Pre values were used as the comparator; thus Pre HipFx values were approximately set to 1. In the case of comparing HipFx samples against CON, the CON values were used as the comparator; thus CON values were approximately set to 1.
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