Chitinase mRNA levels by quantitative PCR using the single standard DNA: acidic mammalian chitinase is a major transcript in the mouse stomach

Abstract

Chitinases hydrolyze the β-1-4 glycosidic bonds of chitin, a major structural component of fungi, crustaceans and insects. Although mammals do not produce chitin or its synthase, they express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). These mammalian chitinases have attracted considerable attention due to their increased expression in individuals with a number of pathological conditions, including Gaucher disease, Alzheimer's disease and asthma. However, the contribution of these enzymes to the pathophysiology of these diseases remains to be determined. The quantification of the Chit1 and AMCase mRNA levels and the comparison of those levels with the levels of well-known reference genes can generate useful and biomedically relevant information. In the beginning, we established a quantitative real-time PCR system that uses standard DNA produced by ligating the cDNA fragments of the target genes. This system enabled us to quantify and compare the expression levels of the chitinases and the reference genes on the same scale. We found that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach. The level of this mRNA in the mouse stomach was 7- to 10-fold higher than the levels of the housekeeping genes and was comparable to that the level of the mRNA for pepsinogen C (progastricsin), a major component of the gastric mucosa. Thus, AMCase mRNA is a major transcript in mouse stomach, suggesting that AMCase functions as a digestive enzyme that breaks down polymeric chitin and as part of the host defense against chitin-containing pathogens in the gastric contents. Our methodology is applicable to the quantification of mRNAs for multiple genes across multiple specimens using the same scale.

Figure 1. Strategy for comparing the gene expression levels of five genes.

The expression levels of the Chit1 and AMCase genes were compared. To evaluate the chitinase levels, we used two housekeeping genes, GAPDH and β-actin, which are constitutively expressed at high levels in most tissues and cells. In addition, we chose pepsinogen C, which is a major component of the gastric mucosa, as a reference gene for the stomach tissues. Using these three reference genes, we evaluated the gene expression levels of Chit1 and AMCase in mouse tissues.

Figure 2. Evaluation of primer suitability for real-time PCR.

PCR primers were selected based on whether they gave one melting temperature (A–E) and a single PCR product on a 10% polyacrylamide gel (F). To verify the specificity of the primers, the dissociation curves of the PCR products for five genes generated using mouse tissue cDNA mixture were examined. The PCR products were analyzed on a 10% polyacrylamide gel, followed by ethidium bromide staining. The nucleotide sequences of the PCR products were confirmed.

Figure 3. Construction of standard template DNA.

A. Schematic representation of the standard template DNA used for real-time PCR. The target fragments of the AMCase, pepsinogen C, Chit1, GAPDH and β-actin cDNAs with flanking sequences and restriction sites were amplified and ligated in a one-to-one ratio into a DNA fragment, which was then cloned into a cloning vector. The linearized standard template DNA was amplified from the plasmid DNA and used as the standard DNA. B. Nucleotide sequence of the standard template DNA. The template DNA, 913 nucleotides long, contained five cDNA fragments (shown in different colors) covering the PCR-target region (shown in bold underline) plus 9–120 bases of the flanking regions and containing restriction sites for BglII, XhoI, PstI, or NotI (bold italic).

Figure 4. Establishment and validation of a quantitative real-time PCR system using the standard template DNA.

The checked DNAs are as follows: A, Chit1; B, AMCase; C, GAPDH; D, β-actin; E, pepsinogen C. (Left) Real-time PCR quantification of each 10-fold serial dilution of the standard template DNA using primer pairs targeting each gene. (Right) The standard curves by using the standard template DNA containing the five cDNA fragments (brown closed circles). In addition, the quantification of full coding cDNAs was carried out using primer pairs for each gene. The target cDNA was amplified from a dilution of the full coding cDNA with a known concentration (see Figure S2) and subsequently analyzed as an unknown sample (blue closed rhombuses). Equal quantities were observed for each tested dilution of the standard curve and full coding cDNA.

Figure 5. Expression of Chit1 and AMCase mRNAs in mouse tissues.

Quantification of Chit1 (A) and AMCase (B) mRNAs in mouse tissues. Both chitinases were quantified by real-time PCR using the standard template DNA. All values obtained were expressed as molecules per 10 ng of total RNA. C. Ratios of AMCase to Chit1. All mRNA copy numbers were derived based on the same standard dilutions. The upper panel indicates the actual number, whereas the lower panel shows the logarithm of the values.

Figure 6. Analysis of Chit1 and AMCase mRNAs and reference gene mRNAs in lung and stomach tissues.

The expression levels of the five genes determined using the cDNAs prepared from lung (A) or stomach (B) tissues from 3-month-old mice (n = 5) were quantified by real-time PCR. The upper panel indicates the actual value, whereas the lower panel shows the logarithm of each value.