housekeeping gene for lymphocyte - (Nov/12/2006 )
We want to detect the mRNA level of a gene only express in the lymphocyte, but, there are many kinds of cells in our sample. So we want to find a reference gene which also only express in lymphocyte. Someone suggest me to use cd3 e or cd8 b as the housekeeping gene. I hope you guys could give me some suggestion. Thanks.
Absolute normalizers standardize the target quantity to the unit amount of sample (ex copies/mL), do not depend on the properties of another sample, it is finding out an intrinsic property of a sample. Using a standard curve, the quantity is interpolated from a range of standards of known concentration. For that you need template of known concentration, perform serial dilutions of this template, assay the unknown sample and standard curve in the same run, and interpolate results
Relative normalizers ensure that the obtained target quantities from equivalent amount of samples are compared. A reference gene is advantageous in cases where the precise quantification of input RNA is not possible (ex in cases where only a small amount of starting template is available). A reference gene is one whose expression level is constant across all test samples and whose expression is not affected by the experimental treatment applied in the study. Use a selection of housekeeping genes (aka internal control genes) with similar quantitative transcription in the tested samples that should not vary among the tissues or cells under investigation. Unfortunately, considerable variability has been reported in the transcription of many housekeeping genes. Keep in mind that any real-time approach is lacking of any meaning (not to mention is disregarded by the specialists) whether it doesn’t prove the constant levels of the normalizers.
Relative normalizers, relative to a reference gene - circumvents the need for accurate quantification and loading of the starting material, convenient with limited starting material, requires the availability of a good reference gene(s) with constant expression in all samples tested and whose expression does not change by the treatment under study. Briefly, one of the sample is chosen as the calibrator (untreated baseline sample), all other samples are expressed as an increase or decrease relative to the calibrator.
The identification of a valid reference for data normalization remains the most stubborn of qPCR problems, none of the solutions proposed are ideal. For most experimental conditions GADPH use is inappropriate and should be discontinued. By contrast, normalization to total cellular RNA advocated as the best method due to the observation that total RNA content revealed relatively little variation in the amount of total RNA per cell (median 2.1 ng/cell). However, normalization to total RNA content requires accurate quantification of the RNA sample. It’s not impossible (methods commonly used: RiboGreen RNA quantification assay www.probes.com; absorbance OD260).
Therefore, I suggest to decide for a selection of commonly tested ref genes and see which suits your experimental conditions, i.e., which have the most stable expression regardless the sample characteristics.
Having all that said, please find below links that you may find useful in your quest
2-microglobulin ( 2m) housekeeping gene was used to normalize the DNA concentration and quality
Selection of reference genes for gene expression studies in human
neutrophils by real-time PCR
Selection of suitable reference genes for accurate normalization of
gene expression profile studies in non-small cell lung cancer
Correlation between apoptosis macroarray gene expression profiling and histopathological lymph node lesions
The following article, “Utility of the Housekeeping Genes 18S rRNA, β-Actin and Glyceraldehyde-3-Phosphate-Dehydrogenase for Normalization in Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction Analysis of Gene Expression in Human T Lymphocytes”, cited by Bustin in
may be also of interest.
It says “The accuracy of 18S rRNA, -actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the expression level of 18S rRNA, -actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T-cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of -actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated TCR+, CD4+ and CD8+ subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h….Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes”.
I appologize if it's not what exactly you are expecting to get