Journal: International Journal of Molecular Sciences

Article Title: The Effect of Tropical Temperatures on the Quality of RNA Extracted from Stabilized Whole-Blood Samples

doi: 10.3390/ijms231810609

Figure Lengend Snippet: Comparison RNA concentration, purity, and integrity between PAXgene ® and Tempus ™ blood collection systems under suboptimal tropical storage conditions. ( A ) Mean RNA yield normalized to whole-blood volume (ng/μL), A260/A280 ratios determined spectrophotometrically, and RNA integrity number (RIN) across the different conditions (Fresh ( n = 8), Day1/Control; immediately frozen at −80 °C for later extraction and various temperatures (25, 30, 35, 40 °C) (matched subjects n = 3)). ( B ) Scatter plots revealed correlations between normalized RNA concentration (ng/uL) by Spectrophotometer or Bioanalyzer for PAXgene ® (left panel) and Tempus ™ (right panel). Pearson’s correlation assessed correlations between variables. ( C ) Electropherograms showed two distinct peaks ( 28S and 18S ), gel images showed two bands comprising the 28S and 18S from high-quality RNA, and smears indicated RNA degradation. The uncropped non-quantitative gel per n is shown; 18S and 28S peaks of sample #3 (Fresh) and #2 (Control, 5 days at 25 °C and 5 days at 40 °C). Note the different scales in C. The dashed line indicates OD260/A280 = 2.0 for high-quality RNA. Effect of tube type in the linear regression models for each temperature point are indicated; *** p < 0.001, ** p < 0.01, * p < 0.05, ns, non-significant; RIN, RNA integrity number.

Article Snippet: Total RNA from whole blood was extracted from PAXgene ® Blood RNA Tubes using MagMAX ™ for Stabilized Blood Tubes RNA Isolation Kit (Life Technologies, CA, USA) according to the manufacturer’s protocol, including a TURBO ™ DNase and protease step.

Techniques: Concentration Assay, Spectrophotometry

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Tropical Temperatures on the Quality of RNA Extracted from Stabilized Whole-Blood Samples

doi: 10.3390/ijms231810609

Figure Lengend Snippet: Cycle thresholds (Ct) of housekeeping genes assessing RNA from PAXgene ® and Tempus ™ stored at different temperatures and times. The mean cycle threshold (Ct) values for 18S , SDHA, and TBP with 100–200 base pair (bp) (short-amplicon) or 200–300 bp (medium-amplicon) lengths across different conditions (control and at multiple storage temperatures and days (matched n = 3)). *** p < 0.001, ** p < 0.01, * p < 0.05; ns, non-significant; †, data from only two observations were potentially available due to LOD. Blue, Tempus ™ Blood RNA tubes; red, PAXgene ® Blood RNA tubes.

Article Snippet: Total RNA from whole blood was extracted from PAXgene ® Blood RNA Tubes using MagMAX ™ for Stabilized Blood Tubes RNA Isolation Kit (Life Technologies, CA, USA) according to the manufacturer’s protocol, including a TURBO ™ DNase and protease step.

Techniques: Amplification

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Tropical Temperatures on the Quality of RNA Extracted from Stabilized Whole-Blood Samples

doi: 10.3390/ijms231810609

Figure Lengend Snippet: Cycle thresholds (Ct) in dependence on amplicon length and RNA integrity (RIN). ( A ) Scatter plots show the Ct values in dependence on amplicon length and RNA integrity (RIN) for different lengths of SDHA amplicons differences for each tube type. ( B ) Spearman correlation of the ΔCt values with RIN value (RNA quality) for medium-length amplicons (200–300 bp) of 18S ( left ), SDHA ( middle ), and TBP ( right ). Blue, Tempus ™ Blood RNA tubes; red, PAXgene ® Blood RNA tubes.

Article Snippet: Total RNA from whole blood was extracted from PAXgene ® Blood RNA Tubes using MagMAX ™ for Stabilized Blood Tubes RNA Isolation Kit (Life Technologies, CA, USA) according to the manufacturer’s protocol, including a TURBO ™ DNase and protease step.

Techniques: Amplification

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Tropical Temperatures on the Quality of RNA Extracted from Stabilized Whole-Blood Samples

doi: 10.3390/ijms231810609

Figure Lengend Snippet: Experimental design. Experiment A. Comparison of total RNA yields, purity, and integrity using spin column-based and magnetic bead-based kits for the two types of blood stabilization systems (PAXgene ® Blood RNA Tubes and Tempus ™ Blood RNA Tubes). All RNA samples were extracted fresh post collection. Experiment B . Systematic testing of different temperatures (25, 30, 35, or 40 °C) and storage times (0, 1, 5, 7, or 10 days) were immediately frozen at −80 °C for later extraction with matched samples ( n = 3). Samples immediately frozen at −80 °C for later extraction is referred to “D1/Control”. Unmatched samples ( n = 8) collected in PAXgene ® or Tempus ™ RNA stabilizing systems and processed post collection immediately at optimal laboratory conditions (D0/Fresh).

Article Snippet: Total RNA from whole blood was extracted from PAXgene ® Blood RNA Tubes using MagMAX ™ for Stabilized Blood Tubes RNA Isolation Kit (Life Technologies, CA, USA) according to the manufacturer’s protocol, including a TURBO ™ DNase and protease step.

Techniques: