RNA Sampling and Diversity of Samples
Supplemental FigureS1:
RNA sampling and diversity of samples
Total RNA was isolated from V. carteri females at different biological or experimental conditions.A, RNA was isolated at several time points throughout the asexual and sexual life cycles. The sexual pathway wasinduced by adding the sex-inducer glycoprotein [1,2,3].Each time point ofRNA isolation is indicated by a vertical bar throughout both asexual (blue) and sexual (red) life cycles. Numbers indicate the developmental stage: 1, release of juveniles; 2, maturation of gonidia; 3, initiation of cleavage divisions; 4, during cleavage divisions; 5, during embryonic inversion (only in sexual cycle) or at the end of embryonic inversion (only in asexual cycle); 6, during final cellular differentiation; 7, expansion of the ECM; 8, further expansion of the ECM; 9,release of juveniles.B, RNA was isolated from different cell types. Isolation of cell types occurred at two different developmental stages[4]: just before initiation of cleavage divisions (see images) and after embryonic inversion. C, RNA was isolated from heat-stressed organisms. The heat shock condition was effected by elevating the culture temperature from 28ºC to 42.5ºC for 100 min and then to 45ºC for 20 min (red thermometer) [5]. The control remained at 28ºC (black thermometer). D, RNA was isolated from cold-stressed organisms. The cold shock condition was effected by reducing the culture temperature from 28ºC to 14ºC for 2 h (blue thermometer)[5]. The control remained at 28ºC (black thermometer). E,RNA was isolated from organisms at high light. The high light condition was caused by increasing the illumination intensity to 450 µmol photons m-2s-1 PAR for 3 h. The control remained at 100 µmol photonsm-2s-1 PAR. F,RNA was isolated from organisms at low light. The low light condition was caused by reducing the illumination intensity to 25 µmol photons m-2s-1 PAR for 3 h. The control remained at 100 µmol photonsm-2s-1 PAR. G, RNA was isolated one hour after wounding. The mechanical wounding of spheroids was caused by using a Dounce homogenizer; in this way spheroids were gently slit into hemispheres [5]. The control remained untreated.
Supplemental Figure S2:
Separate analysis of each sample pool by geNorm – Average expression stability during stepwise exclusion of the least stable candidate reference gene
Stepwise exclusion of the gene with the highest M value allows ranking of the tested candidate reference genes according to their expression stability. Each sample pool was analysed separately using the geNorm algorithm. The cut-off value was 0.5 [6]; M values below this threshold indicate adequate stability in gene expression.
References
[1] A. Hallmann, K. Godl, S. Wenzl, M. Sumper, The highly efficient sex-inducing pheromone system of Volvox, Trends Microbiol 6 (1998) 185-189.
[2] R.C. Starr, L. Jaenicke, Purification and characterization of the hormone initiating sexual morphogenesis in Volvox carteri f. nagariensis Iyengar, Proc Natl Acad Sci U S A 71 (1974) 1050-1054.
[3] H. Tschochner, F. Lottspeich, M. Sumper, The sexual inducer of Volvox carteri: purification, chemical characterization and identification of its gene, EMBO J 6 (1987) 2203-2207.
[4] A. Kianianmomeni, G. Nematollahi, A. Hallmann, A gender-specific retinoblastoma-related protein in Volvox carteri implies a role for the retinoblastoma protein family in sexual development, Plant Cell 20 (2008) 2399-2419.
[5] A. Kianianmomeni, K. Stehfest, G. Nematollahi, P. Hegemann, A. Hallmann, Channelrhodopsins of Volvox carteri are photochromic proteins that are specifically expressed in somatic cells under control of light, temperature, and the sex inducer, Plant Physiol 151 (2009) 347-366.
[6] M. Rocha-Martins, B. Njaine, M.S. Silveira, Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development, PLoS One 7 (2012) e43028.
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