Comprehensive selection of reference genes for quantitative RT-PCR analysis of murine extramedullary hematopoiesis during development

<div><p>The purpose of this study was to perform a comprehensive evaluation and selection of reference genes for the study of extramedullary hematopoiesis during development and the early post-natal period. A total of six candidate reference genes (<i>ACTB</i>, <i>GAPDH</i>, <i>HPRT1</i>, <i>PPID</i>, <i>TBP</i>, <i>TUBB3</i>) in four organs (heart, liver, spleen, and thymus) over five perinatal time points (Embryonic days 14.5, 16.5, 18.5, Post-natal days 0, 21) were evaluated by quantitative real-time PCR. The expression stability of the candidate reference genes were analyzed using geNorm, NormFinder, Bestkeeper, Delta CT method, and RefFinder software packages. Detailed methodology for isolation of high quality/purity RNA and analysis is presented. Detailed analysis demonstrated that <i>TBP</i> is the best single reference gene for embryonic samples and <i>HPRT1</i> is the best single reference gene for post-natal and pooled embryonic and post-natal samples. Organ-level analysis demonstrated that <i>HPRT1</i> was the most suitable reference gene for heart, liver and thymus samples, while <i>TBP</i> was the best candidate for spleen samples. In general, <i>TUBB3</i> was consistently the least stable gene for normalization. This is the first study to describe a systematic comprehensive selection of reference genes for murine extramedullary hematopoietic tissues over a developmental time course. We provide suggested reference genes for individual tissues and developmental stages and propose that a combination of reference genes affords flexibility in experimental design and analysis.</p></div

Reference gene expression stability over time.

<p>Distribution of the raw Cp values for embryonic organs (A), post-natal (B) and pooled (C). The box plot of the real-time PCR compiles 60 raw Cp values for each gene. All three plots demonstrate a similar trend. A highest variation is showed in <i>TUBB3</i> compared with the other genes.</p

PCR efficiency of six reference genes.

<p>PCR efficiency of six reference genes.</p

RNA quality and purity.

<p>RNA Integrity Number (RIN) values obtained from Agilent 2100 Bioanalyzer System was utilized to test the quality of the RNA samples and the Histogram of Frequency Distribution of the A260/A280 ratio was used to indicate the purity of the samples. <b>(A)</b> Electrophoresis of representative sample types allows visual inspection of RNA quality of the different tissues (heart, liver, spleen, and thymus). <b>(B)</b> The electropherogram shows 18S (left) and 28S (right) peaks indicative of RNA integrity of the different organs. <b>(C)</b> Histogram of frequency showing the distribution of the purity of RNA was performed using the ratio of absorbance at A260 nm/absorbance at A280 nm. X axis shows the A260/A280 for the 60 samples tested independently.</p

Ranking of the candidate reference genes by organ type.

<p>Ranking of the candidate reference genes by organ type.</p

Gene expression analysis by organ.

<p>Expression profiles of individual reference genes in each of the tissues (heart, liver, spleen, thymus) using pooled samples representing all time points. Fig shows the mean +/- SEM of 15 samples for each tissue type. * <i>p</i> ≤ 0.05, ** <i>p</i> ≤ 0.01, *** <i>p</i> ≤ 0.001, **** <i>p</i> ≤ 0.0001.</p

Stability analysis of the six reference genes.

<p>geNorm (A-C) and NormFinder (D-F) were used to identify reference genes for normalization. The 1^st row are embryonic organs (A and D), the 2^nd row shows post-natal organs (B and E), and 3^rd row represent pooled organs (C and F). The accumulated SD calculated in NormFinder is shown in the right column.</p