Validating internal controls for quantitative plant gene expression Free webcamsex with a stranger
Data normalization based on reference genes is essential for obtaining reliable results for q RT-PCR assays.This study evaluated potential reference genes of Chinese yam (Dioscorea opposita Thunb.), which is an important tuber crop and medicinal plant in East Asia.Our results provide the foundation for reference gene(s) selection in D.opposita and will contribute toward more accurate gene analysis studies of the genus Dioscorea.
Northern blotting, semiquantitative reverse transcription-PCR, and reverse transcription quantitative real-time PCR (q RT-PCR)  have each been used extensively in modern biological research.The use of reference genes is commonly accepted as the most appropriate normalization strategy .Consequently, the reliability of the quantitative results is highly dependent on the choice of appropriate reference genes for use in normalization.Although it is widely used for gene expression analysis due to these advantages, q RT-PCR suffers from certain pitfalls such as differences in initial sample amount, RNA integrity issues, differences in the efficiency of c DNA synthesis, and differences in the overall transcriptional activity of the tissues or cells analyzed ; all of these factors can make the quantification of gene transcripts unreliable.To avoid bias, the selection of an appropriate normalization method becomes imperative for obtaining reliable quantitative gene expression results.Thus, there is an urgent need to systematically evaluate the stability of potential reference genes for particular experimental systems prior to adopting them for use in q RT-PCR normalization strategies.There have been a number of studies on the validation of reference genes in different plants including model plants (Arabidopsis [16, 17], rice [18, 19], tomato [20, 21], tobacco , etc.), crop plants (soybean [23, 24], pea , sugarcane , coffee [27, 28], peanut , cotton [2, 30], Brassica napus , wheat , etc.), vegetables (potato , chicory , cucumber , pepper , radish , etc.), fruits (berry , peach , banana , apple , etc.), flowers (petunia , rose [39, 40], etc.), tree plants, longan tree , and poplar .The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed.We identified the most stable genes for q RT-PCR studies using combined samples from different organs.The first stage is the explant stage, during which a nodal segment is inoculated and the culture is initiated, named PLBs-0; the second stage is the explant swelling stage; the white small protrusions appear at leaf axils during this stage, named PLBs-I; the third stage is the primary PLB stage; white small protrusions become large and turn into light green PLBs cluster during this stage, named PLBs-II; the 4th stage is the mature PLB stage; the light green PLBs cluster grows up and becomes ivory coloured, named PLBs-III.For PLBs, samples were collected at four different stages.