Analysis of mouse glomerular podocyte mRNA by single-cell reverse transcription-polymerase chain reaction

The polymorphic control region of mitochondrial DNA (mtDNA) is becoming more commonly used in forensic applications to differentiate among individuals in a population. Two hypervariable regions (HV1 and HV2) are often sequenced following amplification of the mtDNA via the polymerase chain reaction (PCR). More rapid screening assays would reduce both the effort and the expense of comparing two samples. A methodology has been developed that first uses restriction endonuclease digestion of the PCR-amplified mtDNA using RsaI and MnlI and then capillary electrophoresis (CE) to separate and size the PCR-RFLP fragments. This rapid procedure offers an alternative method for screening of polymorphisms in amplified mtDNA samples. In addition, the presence of a T-->C transition at position 16189, which gives rise to the so-called "C-stretch" in HV1, may be predicted from the presence of nonspecific PCR products in the CE results.     

Semiquantitative reverse transcription-polymerase chain reaction analysis of mRNA for growth factors and growth factor receptors from normal and healing rabbit medial collateral ligament tissue

By studying western lowland gorillas (Gorilla gorilla gorilla, n = 8) in zoological gardens via ethological and non-invasive physiological techniques, we have demonstrated that their postpartum maternal behavior is related negatively to their postpartum urinary titers of cortisol. On the basis of this finding, it is proposed that postpartum stress contributes to disrupted maternal behavior in the gorilla in captivity. Morning urine samples were collected with a mean sampling interval of 1.6 days from Day 14 prepartum to Day 14 postpartum (n = 11 pregnancies). Creatinine-indexed (Cr) urinary cortisol titers declined significantly between Day 9 to 1 prepartum (0.634 +/- 0.014 microg/mg of Cr, mean +/- SEM) and Day 1 to 6 postpartum (0.396 +/- 0.030 microg/mg of Cr, mean +/- SEM; p < 0.01-0.001). For each pregnancy, the relative postpartum decline in urinary cortisol was calculated as (microg of cortisol/mg of Cr Day 1 to 4)/(microg of cortisol/mg of Cr Day -4 to -1). Values ranged from 0.35 to 1.12, were independent of absolute prepartum cortisol titers, and were interpreted as evidence of inter-female differences in postpartum hypothalamo-pituitary-adrenal axis activity and, therefore, postpartum stress. This postpartum stress index was negatively correlated with the amount of time (0-100%) that females carried and supported their 0-14 day-old infants in a ventral position during locomotion (r(s) = -0.68, p < 0.05) and tended to be negatively correlated with the total amount of time (0-100%) they spent in ventro-ventral contact with their infants (r(s) = -0.58; p < 0.10). This study provides the first physiological evidence that postpartum stress is an important etiologic factor in gorilla maternal failure in captive environments.     

Quantification of androgen receptor mRNA in tissues by competitive co-amplification of a template in reverse transcription-polymerase chain reaction

We describe a polymerase chain reaction (PCR)-based method for the quantification of androgen receptor (AR) mRNA in tissues. The amount of PCR products depends on the exponential amplification of the initial cDNA copy number; therefore minor differences in the efficiency of amplification may dramatically influence the final product yield. To overcome these tube-to-tube differences in reaction efficiency, an internal control AR cRNA was reverse transcribed along with the target mRNA using the same primers. This standard was obtained by deleting a 38 bp fragment from an amplified bovine AR sequence, which was then subcloned and transcribed into cRNA. Known dilutions of the competitor cRNA were spiked into a series of RT-PCR reaction tubes containing equal amounts of the target mRNA. Following RT-PCR, the co-amplified specimens obtained were separated by gel electrophoresis and quantified by densitometric analysis of ethidium bromide stain. We applied this method to quantify the AR-mRNA in skeletal muscle of castrated as well as from intact male cattle. The applicability of the quantification system for AR-mRNA described herein was demonstrated for other species, e.g. man.