Abstract
A method for quantification of mRNA coding for the allergen Lol p 1 is described. Quantitative RT-PCR is performed using a mutant RNA sequence as an internal standard, slightly longer than corresponding wild-type RNA. The mutant, which contains a unique Hind III restriction site, has been synthesised by splice overlap extension PCR of corresponding wild-type cDNA. Purification has been effected by ligation into pCR-Script SK(+) and introduction into E. Coli XL1-MFR. The mutant is obtained by in vitro transcription of purified and BamH I-cut plasmid. RT-PCR is performed in one-tube procedures where RTase and polymerase are both added at the same time, thus minimising the danger of contamination. After digestion with Hind III the product is analysed by polyacrylamide gel electrophoresis in an automated sequencer, AlfExpress. A special software (Fragment manger) allows direct estimation of the areas under respective peaks. A linear correlation between area and DNA amount exists over more than 2-log range. A preliminary ``titration'' to estimate best relation between standard and unknown sample is recommended. Samples comprising 30 pollen grains can be successfully analysed. Standard deviations of about 10% can be obtained in triplicate samples. For increased reliability at least five samples of each batch of pollen should be analysed. The quality of the pollen material should be checked by light microscopy.
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Berggren, B., Källersjö, M. & Suphioglu, C. A method to quantify amounts of mRNA coding for Lol p 1 in pollen from Lolium perenne. Aerobiologia 16, 261–270 (2000). https://doi.org/10.1023/A:1007606125930
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DOI: https://doi.org/10.1023/A:1007606125930