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

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

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

PCR efficiency of six reference genes.

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

37/67-laminin receptor facilitates neural crest cell migration during enteric nervous system development

Enteric nervous system (ENS) development is governed by interactions between neural crest cells (NCC) and the extracellular matrix (ECM). Hirschsprung disease (HSCR) results from incomplete NCC migration and failure to form an appropriate ENS. Prior studies implicate abnormal ECM in NCC migration failure. We performed a comparative microarray of the embryonic distal hindgut of wild-type and EdnrBNCC−/− mice that model HSCR and identified laminin-β1 as upregulated in EdnrBNCC−/− colon. We identified decreased expression of 37/67 kDa laminin receptor (LAMR), which binds laminin-β1, in human HSCR myenteric plexus and EdnrBNCC−/− NCC. Using a combination of in vitro gut slice cultures and ex vivo organ cultures, we determined the mechanistic role of LAMR in NCC migration. We found that enteric NCC express LAMR, which is downregulated in human and murine HSCR. Binding of LAMR by the laminin-β1 analog YIGSR promotes NCC migration. Silencing of LAMR abrogated these effects. Finally, applying YIGSR to E13.5 EdnrBNCC−/− colon explants resulted in 80%-100% colonization of the hindgut. This study adds LAMR to the large list of receptors through which NCC interact with their environment during ENS development. These results should be used to inform ongoing integrative, regenerative medicine approaches to HSCR

Enterocolitis Score by Anatomic Location.

<p>The distal small intestine, proximal colon and mid-colon of P21-24 and P26-29 <i>EdnrB</i>^{<b>NCC+/-</b>} and <i>EdnrB</i>^{<b>NCC-/-</b>} animals were analyzed using the previously published Murine Enterocolitis Grading System. No inflammation was seen at the P21-24 time point or in the distal small intestine in either group. Data are presented for proximal colon and mid-colon at P26-29, showing depth of inflammation and severity of inflammation on an ordinal scale.</p><p>Statistically significant <i>p</i> values are indicated by an *.</p><p>Enterocolitis Score by Anatomic Location.</p