The overall goal of this procedure is to analyze the expression levels of histone H one subtypes through both the analysis of H one mRNA levels and the measurement of H one protein levels to accomplish mRNA analysis, total RNA is first extracted from cultured cells or mouse tissue. The second step of the procedure is to perform a reverse transcription of the total RNA with random primers, followed by quantitative real-time PCR assays for each of the H one subtype genes. Hisone H one protein analysis begins with preparation of total hisone extracts from mouse tissues or cultured cells.
This is followed by fractionation and quantification of each H one subtype and core hisone H two B by reverse phase HPLC analysis. Ultimately quantitative real-time PCR and reverse phase HPLC analysis show expression levels of both mRNA and proteins. For each H one subtype.
We are graduate students of Dr.Yuhan FES lab at School of Biology of Georgia Institute of Technology. Today we'll show you a set of comprehensive assays for analysis of expression of H ones in mammalian H one subtypes. Through these assays, you can get a quantitative measurement of expression levels of both mRNA and protein in various H one subtypes.
Histone H one is the major chromo structural protein that facilitates higher auto chromo folding multiple histone H one variants existing memos that are differentially expressed during cell differentiation and development expression. Profiling of these H one subtypes is necessary for better understanding of regulation of higher auto chromo structure and function. So let's get started.
Before RNA extraction, all working surfaces and pipettes should be wiped with 70%ethanol and treated with RNAs decontamination solution. This practice reduces the chances of RNAs contamination and RNA degradation. In addition, gloves should be worn for all procedures to begin extraction of RNA from adherent cell culture, aspirate culture medium, and rinse the cells with PBS.
Then remove the PBS from the wells. Sufficient triol reagent is necessary for obtaining high quality RNA. Use one milliliter triol reagent to extract RNA from one well of a six well plate proceed to extract RNA from cells according to the manufacturer's manual for triol reagent following RNA extraction measure RNA concentration using the NanoDrop 1000.
The typical yield ranges from five to 15 micrograms of RNA per one times 10 to the sixth cultured cells. Before proceeding to CDNA synthesis, analyze RNA quality by GE electrophoresis to eliminate the potential contamination of the RNA with trace amounts of genomic DNA treat RNA samples with RNA free DN per manufacturer's instruction for reverse transcription of total RNA into CD NA.Utilize the superscript three first strand synthesis system according to the manufacturer's instructions. Since Mr.mRNA of most H one genes lack poly A tales, it is critical to use random hexamer instead of oligo DT as primers.
For CDNA synthesis, following reverse transcription CD NA amplification can be achieved through Q-R-T-P-C-R as detected by cyber green dye prior to the Q-R-T-P-C-R assay. Forward and reverse PCR primers specific for each H one gene and the internal reference gene were designed and the primer efficiency was tested by standard curve method as described in the written protocol. Proceed to prepare each PCR reaction by mixing primers CDNA and CYBERG green super.
Mix the reactions well in a micros EAL 96, well PCR plate use micros EAL B adhesive seals to ensure that the plate cover is sealed to the plate. Then vortex a sealed plate and briefly spin down the reaction mixtures. Place the plate in the My IQ single color real-time PCR detection system for QPCR.
Set up the QPCR conditions and perform the run as instructed in the text following amplification. Examine the amplification curves for PCR efficiency and threshold of cycle or CT values. The threshold line can be automatically set by the IQ five optical system software version 2.0.
Because cyber green detects any double stranded DNA, it is critical to perform a melting curve run following the QPCR to ensure that the desired amplicon, but not primer, dimers or contaminants are amplified and detected. For melt curve analysis program, the QPCR instrument to heat the samples from 55 degrees centigrade to 95 degrees centigrade in 0.5 degrees centigrade increments with data collection. Since temperature or TM of double stranded, DNA is dependent on amplicon length and GC content, different amplicons will have different tms.
Examine the derivative melting curves of the QPCR products to confirm the specific melting temperature of desired amplicons, as well as the lack of noise amplicon peaks, such as the melting curves of H one A PCR amplicons shown here with a TM of 86 degrees centigrade. Proceed to analyze QPCR data with IQ five optical system software. Normalize the expression values of H one isoform genes with the expression of housekeeping genes to obtain relative expression levels of H one genes.
After dissecting the organ of interest from a euthanized mouse, rinse it with ice cold PBS. In this demonstration preparation of total, his stones is performed on the liver following wash, mince the tissue into pieces with a razor blade. Transfer the minced tissue to a down homogenizer then adds sucrose buffer to the homogenizer at a volume of 10 milliliters per gram of tissue.
Proceed to homogenize the tissue with 10 to 15 strokes After transferring the homogenates to a 15 milliliter tube, perform centrifugation of the samples at 500 RPM for 30 seconds. Following centrifugation. Carefully transfer the supernatant to a new tube and centrifuge at 2000 RPM for five minutes.
To pellet the cells resuspend the cell pellet in 10 milliliters of sucrose buffer, supplemented with 0.5%NP 40. Transfer the sample to a down homogenizer and down 10 strokes within a 20 minute incubation. At this point, nuclei are obtained.
Examine the nuclei quality under a microscope. After pelleting the nuclei by centrifugation at 2000 RPM for five minutes, the supernatant can be discarded. Next, resuspend the nuclei pellet in three milliliters of high salt buffer per one gram of tissue.
Transfer the sample to a small down homogenizer and homogenize with five to 10 strokes. Eloqua the suspension into three einor tubes. Incubate the tubes on ice for 20 minutes.
Follow incubation with centrifugation at 14, 000 RPM for 10 minutes to pellet chromatin. After discarding the supernatant extract total histones by adding 0.8 milliliters of 0.2 normal sulfuric acid to each chromatin pellet. Use an einor tube pestle downs to grind the pellet well until the pellet is completely dissociated.
Incubate the samples on a rotating platform at four degrees centigrade overnight. Subsequent to centrifugation, transfer the supernatant into two EOR tubes. Add 2.5 volumes of ice cold ethanol to each tube.
Keep the samples at negative 20 degrees centigrade overnight the following day, centrifuge at 14, 000 RPM for 10 minutes to pellet total histones and discard the supernatant. After washing the pellet three times with 70%ethanol, leave the open tubes on the bench for 20 to 30 minutes to air dry and proceed to HPLC analysis.Immediately. Resus, suspend the histone pellet in the recommended volume of double distilled water depending on the capacity of the reverse phase column and the HPLC instrument centrifuge at 14, 000 RPM for five minutes.
To remove insoluble residues, inject 50 to 100 micrograms of total histone protein onto the reverse phase column of the HPLC system, such as the GE ACTA purifier UPC 900.Loading. An excess amount of protein should be avoided to prevent clogging of the column fraction eight, the linker, histones and core histones with an increasing aceto nitrile gradient as listed in the written protocol, the effluent is monitored at 214 nanometers and the HPLC profiles are recorded and analyzed. The protein fractions can also be collected with the GE FRAC nine 20 fraction auto collector for further analysis.
Shown here are typical amplification curves of H one A-Q-P-C-R reactions using CDNA prepared from mouse, liver, and mouse embryonic stem cells. Evaluation of the amplification plot shows that the triplicate QPCR reactions of each sample gave consistent signals with almost identical CT values suggesting high reproducibility. The lack of buildup of amplicons from RT minus QPCR reactions indicates that genomic DNA contamination was not present or minimal utilizing the CT values of H one genes and housekeeping genes, the relative RNA expression levels of each H one gene was calculated.
The graphs shown here are examples of calculated results for H one A and H one zero genes. The relative expression levels of H one A mRNA are higher in mouse embryonic stem cells as compared with mouse liver, whereas H one zero expression is much higher in liver than in the embryonic stem cells. The difference in expression of H one A or H one zero in mouse embryonic stem cells versus adult mouse liver is also evident from HPLC profiles of histone proteins.
H one zero. The differentiation specific H one is accumulated to a large amount in the adult mouse liver. In contrast, H one zero protein is nearly absent in undifferentiated mouse embryonic stem cells.
On the other hand, H one A is highly expressed in the embryonic stem cells, but presents in low levels in adult mouse liver through quantification of H one peaks. In the HPLC profile, the relative proportion of each individual H one subtype within the H one family is determined in adult mouse liver. Furthermore, the values of individual H one subtype per nucleosome can be calculated from the ratio of the normalized peak value for the absorbance at 214 nanometers of corresponding H one subtypes to one half of the normalized value for H two B.This value can similarly be calculated for total H one per nucleosome.
Once mastered the Q-R-T-P-C-R analysis of H one subtype can be done in one day and histo extraction and HPRC analysis can be done in three days if it performed properly. While attempting Q-R-T-P-C-R analysis of histone H one genes, it's critical to avoid RNA degradation and eliminate the genomic DNA contamination, and most importantly to include random primers into reverse transcription reaction. During histone extraction and HPOC analysis, it's important to add protein inhibitors freshly to the extraction buffer to avoid protein degradation, and also we have to pay an attention to fully disaggregate chromatin to facilitate the complete his extraction.
After watching this video, you should have a good understanding of how to comprehensively analyze the expression of mammalian histo H one subtypes at both mRNA and protein levels.
