Real-time PCR quantification of human complement C4A and C4B genes

Abstract

Background: The fourth component of human complement (C4), an essential factor of the innate immunity, is represented as two isoforms (C4A and C4B) in the genome. Although these genes differ only in 5 nucleotides, the encoded C4A and C4B proteins are functionally different. Based on phenotypic determination, unbalanced production of C4A and C4B is associated with several diseases, such as systemic lupus erythematosus, type 1 diabetes, several autoimmune diseases, moreover with higher morbidity and mortality of myocardial infarction and increased susceptibility for bacterial infections. Despite of this major clinical relevance, only low throughput, time and labor intensive methods have been used so far for the quantification of C4A and C4B genes.

The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor

“…This allowed us to evaluate the individual SB transposase's enzymatic activities and adjust for possible difference in electroporation efficacy between different samples. Measurement of RPPH1, a subunit of RNase P (21), which is present at one copy on chromosome 14q11.2, was used as internal control in real-time Q-PCR for both quantification of excision circles and transposon DNA species. By varying the relative amounts of DNA plasmids coding for the transposases, SB100X reached peak activity at a concentration of 5 μg/electroporation (Figure 2b).…”

Common Genetic Variants of the Human Steroid 21-Hydroxylase Gene (CYP21A2) Are Related to Differences in Circulating Hormone Levels

“…Briefly, C4 -type-specific quantitative PCRs (qPCR) [23], and CYP21 -gene-specific qPCR [19] were applied to determine gene copy numbers. Haplotypic or genotypic nested polymerase chain reaction (PCR) products of CYP21A2 were generated from the templates of allele-specific long-range PCRs [4], then CYP21A2 intron 2 was sequenced by SEQ_12F_v2 and SEQ_16R sequencing primers [19].…”

Polymorphisms of Beta Defensins Are Associated with the Risk of Severe Acute Pancreatitis

“…To amplify DEFB4 and albumin in a one-tube biplex assay, limiting primer conditions were identified to avoid competition of the two reactions. Quantification was performed by the comparative CT (threshold cycle) method, as described previously [25]. …”