The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models

Medhurst, Andrew D.; Harrison, David; Read, Simon; Campbell, C. A.; Robbins, Melanie J.; Pangalos, Menelas N.

doi:10.1016/s0165-0270(00)00178-3

Cited by 241 publications

(157 citation statements)

References 15 publications

Supporting

Mentioning

154

Contrasting

Unclassified

Order By: Relevance

“…The cDNA used to generate the relative standard curves was from the ST muscle of a barrow not included in the study and slaughtered at a similar age and BW as the experimental animals. When PCR amplification is maximally efficient, resulting in a doubling of product in every cycle, the slope will be −3.3 (Medhurst et al, 2000). In the present study, the mean slope of the six genes was − 3.37 ± 0.25.…”

Section: Experimental Design Animals and Feedingmentioning

confidence: 43%

Compensatory growth feeding strategy does not overcome negative effects on growth and carcass composition of low birth weight pigs

Madsen

Bee

2015

Animal

View full text Add to dashboard Cite

The aim of this study was to evaluate whether the compensatory growth feeding strategy could be a suitable solution for overcoming the negative effects on growth, carcass composition and meat quality of low birth weight pigs. Forty-two Swiss Large White barrows from 21 litters were selected at weaning and categorized into either being light (L; > 0.8 and < 1.3 kg) or heavy (H; > 1.7 kg) birth weight pigs. From 27.8 kg BW, pigs were assigned within birth weight group to one of three feeding groups: AA: ad libitum access to the grower and finisher diet, RR: restricted access to the grower and finisher diet or RA: restricted access to the grower diet and ad libitum access to the finisher diet. At slaughter, the longissimus (LM) and semitendinosus (STM) muscles were removed from the right side of the carcass. Weight, girth and length of the STM and the LM area were determined after muscle excision. Carcass characteristics and meat quality traits were assessed. Using mATPase histochemistry, myofibre size and myofibre type distribution were determined in the LM and STM. Because of longer days on feed, total feed intake was greater (P < 0.01) and feed efficiency was lower (P < 0.01) in L than H barrows. Regardless of the birth weight group, AA and RA barrows grew faster (P < 0.05) than RR barrows. During the compensatory growth period, RA barrows grew faster (P < 0.05) than AA or RR barrows. Growth efficiency did not differ between RA and RR barrows but was greater (P < 0.05) compared with AA barrows. Carcasses of L barrows were fatter as indicated by the lower (P ⩽ ≤ 0.05) lean meat and greater (P ⩽ 0.02) omental and subcutaneous fat percentage. Lean meat percentage was lower (P ⩽ 0.05) in AA and RA than RR barrows. These differences caused by ad libitum feed access tended to be greater (feeding regime × birth weight group interaction; P < 0.08) in L than H barrows. In L barrows, slow oxidative, fast oxidative glycolytic and overall average myofibre size of the LM and the fast glycolytic myofibres and overall average myofibre size of the dark portion of the STM were larger (P ⩽ 0.03) than in H barrows. The study revealed that the compensatory growth feeding strategy was inadequate in overcoming the disadvantages of low birth weight.

show abstract

Section: Experimental Design Animals and Feedingmentioning

confidence: 43%

Compensatory growth feeding strategy does not overcome negative effects on growth and carcass composition of low birth weight pigs

Madsen

Bee

2015

Animal

View full text Add to dashboard Cite

show abstract

“…To confirm absence of genomic contamination, the PCR products were run on an agarose gel to confirm that the amplified product had the proper size (amplified product for apoD: 76 bp). The forward and reverse primers and TaqMan probe for GAPDH was from the article (Medhurst et al, 2000) and the following sequences were used for apoD, FW: 5 0 -CGGTGGCAT CAACGAGAAG-3 0 , RV: 5 0 -TTGAGGGTGAAGCCAAACA GA-3 0 and TaqMan probe: 5-0 FAM-TCCAGCTTGGCTGGC TCTGACATGTT-TAMRA-3 0 . The TaqMan probes for apoD and GAPDH were synthesized with the fluorescent reporter dye FAM (6-carboxy-fluorescein) attached to the 5 0 end and a quencher dye TAMRA (6-carboxy-tetramethyl-rhodamine) to the 3 0 end.…”

Section: Real-time Quantitative Polymerase Chain Reaction Assaymentioning

confidence: 99%

Apolipoprotein D is Elevated in Oligodendrocytes in the Peri-Infarct Region after Experimental Stroke: Influence of Enriched Environment

Rickhag

Deierborg

Patel

et al. 2007

J Cereb Blood Flow Metab

View full text Add to dashboard Cite

Injury to the brain (e.g., stroke) results in a disruption of neuronal connectivity and loss of fundamental sensori-motor functions. The subsequent recovery of certain functions involves structural rearrangements in areas adjacent to the infarct. This remodeling of the injured brain requires trafficking of macromolecular components including cholesterol and phospholipids, a transport carried out by apolipoproteins including apolipoprotein D (apoD). We investigated the changes in the levels of apoD mRNA and protein, and its cellular localization during a recovery period up to 30 days after experimental stroke in the rat brain. In the core of the brain infarct, apoD immunoreactivity but not mRNA increased in dying pyramidal neurons, indicative of cellular redistribution of lipids. During 2 to 7 days of recovery after stroke, the apoD levels increased in the peri-infarct and white matter areas in cells identified as mature oligodendrocytes. The apoD expressing cells were conspicuously located along the rim of the infarct, suggesting a role for apoD in tissue repair. Furthermore, housing animals in an enriched environment improved sensori-motor function and increased the apoD levels. Our data strongly suggest that apoD is involved in regenerative processes and scar formation in the peri-infarct area presumably by enhancing lipid trafficking.

show abstract

“…The sequences of forward, reverse primers and probes, designed by Integrated DNA Technologies (Coralville, IA) were: MAG: probe 5′-AGCCACCGCGTTGAAGCTG TCTGT-3′, forward: 5′-TGTGTAGCTGAGAAGGAGTAT GG-3′, reverse: 5′-ACAGTGCGATTCCAGAAGGATTAT-3′. An oligonucleotide probe (5′-CCGACTCTTGCCCTTC GAAC-3′) specific for the rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control to standardize the amount of sample RNA (Medhurst et al, 2000). For quantification of Real-time RT-PCR results, fluorescent signal intensities were plotted against the number of PCR cycles on a semilogaritmic scale.…”

Section: Real-time Reverse Transcriptase-polymerase Chain Reactionmentioning

confidence: 99%

Exercise decreases myelin‐associated glycoprotein expression in the spinal cord and positively modulates neuronal growth

Ghiani

Ying

Vellis

et al. 2007

Glia

View full text Add to dashboard Cite

To successfully grow, neurons need to overcome the effects of hostile environments, such as the inhibitory action of myelin. We have evaluated the potential of exercise to overcome the intrinsic limitation of the central nervous system for axonal growth. In line with the demonstrated ability of exercise to increase the regenerative potential of neurons, here we show that exercise reduces the inhibitory capacity of myelin. Cortical neurons grown on myelin from exercised rats showed a more pronounced neurite extension compared with neurons grown on poly-D-lysine, or on myelin extracted from sedentary animals. The activity of cyclin-dependent kinase 5, a kinase involved in neurite outgrowth, was found to be increased in cortical neurons grown on exercise-myelin and in the lumbar spinal cord enlargement of exercised animals. Exercise significantly decreased the levels of myelinassociated glycoprotein (MAG), a potent axonal growth inhibitor, suggesting that downregulation of MAG is part of the mechanism through which exercise reduces growth inhibition. It is known that exercise elevates brain-derived neurotrophic factor (BDNF) spinal cord levels and that BDNF acts to overcome the inhibitory effects of myelin. Accordingly, we blocked the action of BDNF during exercise, which suppressed the exercise-related MAG decrease. Protein kinase A (PKA) has been related to the ability of BDNF to overcome growth inhibition; in agreement, we found that exercise increased PKA levels and this effect was reverted by blocking BDNF. Overall, these results show that exercise promotes a permissive cellular environment for axonal growth in the adult spinal cord requiring BDNF action.

show abstract

The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models

Cited by 241 publications

References 15 publications

Compensatory growth feeding strategy does not overcome negative effects on growth and carcass composition of low birth weight pigs

Compensatory growth feeding strategy does not overcome negative effects on growth and carcass composition of low birth weight pigs

Apolipoprotein D is Elevated in Oligodendrocytes in the Peri-Infarct Region after Experimental Stroke: Influence of Enriched Environment

Exercise decreases myelin‐associated glycoprotein expression in the spinal cord and positively modulates neuronal growth

Contact Info

Product

Resources

About