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1、thawed in a 37C water bath, diluted in warm IMDM media containing10% FBS, washed (2) with IMDM, and then pelleted for RNA isolation.Total RNA was isolated by the guanidinium thiocyanate/acid phenolmethod using Trizol reagent in accordance with the manufacturersstandard method. RNA pellets were washe
2、d with 70% ethanol containingdiethyl polycarbonate (DEPC), air dried, and redissolved in 0.01% DEPCby heating at 55C. The integrity and purity of the RNA was assessed byagarose gel electrophoresis. Following the quality assessment of the RNA,aliquots were quantitated by absorbance at 260 and 280 nm.
3、 6. ThemRNA was transcribed into cDNA by using oligo primer .The variableHOXB6 gene was chosen to amplify in vitro real time fluorogenicquantitativeinstrumeny with gene specific peimer by FluorogenicQuantitative Reverse Transcription Polymerase Chain Reaction(FQ-RT-PCR). FQ-RT-PCR was carried out us
4、ing TaqMan probe-basedchemistry. This chemistry provides for a high level of specificity throughthe design of complementary oligonucleotide primers and 5-reporter/3quencher fluorogenic probes. During the normal PCR process thefluorescent probe is cleaved by the native 5-exonuclease activity of TAQpo
5、lymerase that releases the reporter from the quencher, resulting in thegeneration of a sequence-specific signal. The grading diluted positivereserve transcribed production of HOXB6 gene and gluceraldehydephosphate dehydrogenase (GAPDH) gene was used to facture theirstandstandard curves.The threshold
6、 of each sample were recorded andused to calculate the incept cDNA template.Each additional cycle resultsin the release of reporter molecules from the respective probes. Thefluorescence intensity is related to the initial number of RNA copies,which can be assessed by determining the threshold cycle
7、(CT). AllH O X-specific primers and probes were designed againstGenBank-published sequences in association with Primer Express(Applied Biosystems). Endogenous controls were purchased asRNA-specific Pre Developed Assay Reagents.7. Amplifications wereperformed following an initial 2-minute incubation
8、at 55C to allowuracil-N-glycosylase (UNG) to destroy any contaminating RNA,followed by treatment at 94C for 10 minutes to inactivate the UNGenzyme and activate the DNA polymerase. This was followed by setedcycles. An FTC 1000 Sequence Detection System equipped with a96-well thermal cycler was used f
9、or the amplifications. Data werecollected and analyzed with Sequence Detector v1.6.3 software (AppliedBiosystems). 8. The results were showed by means plus or substractingstandard deviation we compared means between groups by LSD .Allthese accomplishtics software spss 14.0. Results : 1 . the mRNAexp
10、ression of HOXB6 gene in the two groups was detected by real timefluorogenic quantitive reserve transcription polymerize reaction andstated by SPSS 14.0 for windows in One Way Aonva and multiplecomparisons. 2. Homeobox genes (HOX) do have a regulatory function inthe differentiation process of hemato
11、poiesis. During the differentiationand proliferation of hematopoietic stem-progenitor cell to colony formingunit-granulocyte-monocyte or erythroid progenitor、colony forming unite-T-lymphocyt invitro, the expression of HOX B6 was significant positive. 3. Comparedwith the expression of CFU-GM and CFU-
12、TL HOXB6 on day 3, thequantity of HOXB6 was obviously higher on day 7 and lower on day 12respectively in each group. Compared with the expression of CFU-EHOXB6 on day 3, the quantity of HOXB6 was obviously higher on day12 in each group. 4. The proliferation progress affected by ATRA.Compared with th
13、e expression of HOXB6 on of normal group, theexpression of HOXB6 of the group ATRA were up-regulated remarkable(p0.05). Total RNA electrophoresed by 1% formaldehydedenaturalized agarose gel showed that the 5S,18S,28S RNA strips wereall explicit and intact, and had no obvious degradation. 6.The cDNA
14、ofthe HOXB6 and the GAPDH gene were reservedly transcribed byRT-PCR,and compared with the standard DNA Marker they were 119bpand 141 bp long respectively. 7.Fluorogenic quantitative reservetranscription polymerize chain reaction(FQ-RT-PCR)method is perfectmethod that can quantify or determine nature
15、 to investigate the HOXgene expression of Erythroid progenitor cells. It has many merits, itsdetecion extention wide, sensitivity high, difinition high and has not PCRapproach, so it avoid polluting each other, the method can poduce moreRNA and make repeating detection.In a word,the method is credibletechnology to investigate the mRNA expression nowaday. Conclusion:1.Homeobox genes (HOX) do ha
