All 12 procedures were carried out for triplicate aliquots, and after storage for one day Run 1 and for 83 days Run 2. The PBMCs were stored in five different storage media, whereby each of the five batches was divided into different aliquots prior to storage, in order to minimize the number of freeze-thaw cycles.
The five batches were aliquoted in numbers and volumes that were appropriate for each protocol. Each RNA extraction kit was carried out in triplicate, i. Removal of RNL is needed to avoid dilution of and interference with the cognate lysis buffers, in order to maximize cell lysis.
For the EM RNA extraction kit, the manufacturer advises to start the extraction on the apparatus in 2-ml volumes. Thereafter, the 12 one-ml aliquots, i. The DNase inactivation reagent was pelleted by centrifugation for 1 min at 20, g , and the supernatant was transferred to a new RNase-free tube. The final volume after DNase treatment and inactivation was RNA purity was assessed by determination of the ratio for absorbance at nm vs. Results were analyzed with the standard LightCycler Software, version 1.
We aimed to assess which part of the RNA extraction procedure i. More detailed electropherograms and gel images of Run 1 and Run 2 are shown in S1 and S2 Figs, respectively. The RNA measurements with Nanodrop were 1. Despite these absolute differences, we observed a strong congruence. Run 1. Storage of PBMCs for one day. Run 2. Storage of PBMCs for 83 days. Error bars represent the standard deviations of results from three independent extractions.
Results are means and standards deviation in ng from three independent extractions. Given the strong congruence between both analysis methods and taking into consideration that the higher values reported by NanoDrop probably resulted from the fact that this method also measured oligo nucleotides, further comparisons were made between procedures and runs only on the basis of Bioanalyzer results. For this purpose, the combinations of each of the storage media with each of the RNA extraction kits was compared Fig 2.
These results were in agreement with the high RNA concentration, as determined by the Bioanalyzer. Results are means and standards deviation from three independent extractions, in ng. Reliable quantification of gene expression is greatly affected by the quality and quantity of the extracted RNA. Therefore, the way samples are stored preceding RNA extraction as well as the kit of choice to extract RNA are crucial.
Thus far, only a limited number of studies compared storage and extraction of RNA from human samples in a methodological manner [ 19 , 23 , 24 ]. RNA integrity and purity must be considered in order to obtain reliable quantification of gene expression [ 25 ]. Both approaches have been shown to be equivalent [ 26 ]. Both NanoDrop and the Bioanalyzer correlated well with regard to the relative values obtained for the different procedures that were compared.
This is in agreement with the results of [ 43 ], reporting RNA concentrations that were 2. This may be because NanoDrop also measures separate nucleotides. In addition, the very similar RNA yields, obtained after separate runs of RNA extraction after one day and after 83 days of storage of PBMCs, lends strong support for the reproducibility of our results. Together, the results suggest that the optimal storage medium also depends on the subsequent RNA extraction kit.
Indeed, according to Qiagen guidelines, RNAlater is not recommended for storage of animal cells—although this is not mentioned in the guidelines of Thermo Fisher Scientific, for the brand of RNAlater that we used. This would indicate that some cells are not precipitated during centrifugation of RNL and that cellular RNA is lost as well, not due to lysis of cells during storage, but due to incomplete recuperation of cells into the RNLp during centrifugation.
However, because neither study indicated the number of cells that were used for RNA extraction, direct comparison is not possible. This result is in agreement with our study in which we obtained between and ng RNA using different RNA extraction kits and storage conditions.
To assess which part of the procedure was most important to improve the RNA yield, i. The results indicated that the RNA extraction kit has the greatest influence on RNA yield, because whenever RP was used, the highest yield was obtained.
Storage in the lysis buffer EML vs. In addition, frozen samples of mammalian cells are preferably stored in the cognate lysis buffer of the RNA extraction kit.
On the other hand, we recently reported that using RNL for the storage of yeast cells gave excellent results [ 31 ], which might be explained because of a better precipitation of yeast cells during centrifugation of RNL, compared to mammalian cells. Our results are supported not only by the reproducibility of the experiments in which two different runs of extractions were carried out, but also by RT-qPCR results. All kits were carried out in triplicate for each of two extraction runs, i.
B: To add up to a starting volume of 2 ml, as prescribed by the manufacturer. D: No addition of cognate lysis buffer, because already added prior to storage. The RIN value is reported on a scale of 1 to 10, whereby values above 7 are considered to represent high quality and non-degraded RNA.
National Center for Biotechnology Information , U. PLoS One. Published online Feb Jonas D. Van Belleghem. Jeffrey Chalmers, Editor. Centrifuge at maximal speed 14, rpm for 20 min. If a low yield is expected, centrifuge for 30 min. Place samples on ice.
There should be a pellet barely visible at the base of each tube. Mix gently. Recentrifuge at 9, rpm for 5 min. Pour off the ethanol and let the pellets air-dry. This is a critical step; if the pellets dry out too much, the RNA crystallizes and is very difficult to resolubilize. If not enough of the ethanol evaporates, this also prevents the RNA from going into solution.
To quicken the evaporation, centrifuge the tubes briefly to force remaining fluid on the side of the tube to the bottom, then pipette off as much of the ethanol as is feasible. The best time to add DEPC treated water to the RNA pellet is when there is only a tiny meniscus of solution left around the pellet itself. Then measure the absorbance at nm.
If less than 1. The successful extraction of short and long RNA relies on the separation from DNA, proteins and cellular debris contained in cell lysate. Next generation sequencing is another method to quantify RNA, now commonly used to identify molecular signatures of tissues or individual cells and assess differential expression Stark et al.
RNA can be purified from lysed cells by mainly two methods: acid guanidinium thiocyanate-phenol-chloroform AGPC and silica-based extraction columns Chomczynski and Sacchi, ; Boom et al. Its low cost, simplicity and adaptability to various biological material make it the most popular method in basic research. However, with this method, RNA purity and quality largely depend on the expertise of the experimenter and on sample handling.
Silica-based columns allow nucleic acids extraction by binding to silica in the presence of chaotropic salts. It is commonly used in commercially available RNA extraction kits such as Qiagen RNAeasy and is amenable to automation for high throughput. Silica-based columns preferentially capture nucleic acids longer than nucleotides but provide poor recovery of short RNA because short RNA tightly bind with silica and are less likely to elute Ali et al.
In this study, we report that some cells, particularly mouse sperm cells, are resistant to commercially available AGPC and lysis solutions used for silica-based columns. Sperm cells cannot be properly lysed, which results in poor RNA yield and significant sample loss.
We observed that reduction of protein disulfide bonds is necessary for the lysis of sperm heads, and that reduction efficiency is pH-dependent, potentially explaining the difference in sperm lysis efficiency of various solutions. Mice were housed in groups of 3—5 animals in individually ventilated cages. Cages were changed weekly. Epididymidis from both sides was incised by several cuts with a fine scissor and placed in 2 ml M2 medium M ml, Sigma-Aldrich. The supernatant was again collected, mixed with 1 ml of somatic cell lysis buffer 0.
Sperm samples were either resuspended in lysis buffer by passage through a 30 G syringe or in the presence of 0. Since the lysis of sperm cells depends primarily on chemical lysis, we recommend future users a simple homogenization to sufficiently break up the cell pellet to obtain a single-cell suspension using 5 mm steel beads for 2 min , Qiagen.
We did not observe any differences between sperm lysis using small or large steel beads. Samples were centrifuged at 12, rcf for 15 min. After an incubation at room temperature for 10 min, samples were centrifuged at 12, rcf for 10 min. After a final centrifugation at 7, rcf for 5 min, the supernatant was carefully removed. DNA content was quantified by fluorometry with the Qubit double-strand DNA high sensitivity assay Q, Invitrogen according to manufacturer instructions.
A testis sample without GoScript reverse transcriptase noRT and a sample without RNA no Input was processed in parallel and served as negative controls. PCR products size was assessed by agarose gel electrophoresis. All samples were run in triplicate.
Melt curve analysis confirmed amplification of single products for each primer. The reduction of disulfide bonds was performed as described previously Han and Han, The solution was briefly mixed by pipetting and absorption measurements started 10 s after DTDP addition. Graphs and statistics were prepared using the software GraphPad Prism 9.
Chemical reactions were drawn in ChemDraw A sperm cell is composed of a head containing a nucleus that carries the paternal genome and RNA, and a flagellum prolonging the head through a mitochondria-rich midpiece, that provides motility Figure 1A. We incubated mouse sperm cells at room temperature in Trizol, a commercially available AGPC RNA extraction reagent containing guanidinium thiocyanate as chaotropic agent. To separate sperm heads from the midpiece and flagellum, and dissociate cell clumps, we passed the sperm samples 10 times through a 30 G needle at the beginning of Trizol incubation.
Despite this treatment, sperm heads remained intact after 5 min of incubation, indicating inefficient lysis Figure 1A. We repeated the procedure and made it more stringent by using longer incubation 30 min in Trizol and strong homogenization with small steel beads at 20 Hz for 10 min. However, intact sperm heads still remained visible Figure 1A. These results confirm previous observations that 2 M guanidinium does not lyse sperm heads Flaherty and Breed, Figure 1.
Mouse sperm cells require reducing agents for lysis in chaotropic solutions. Lysis is not improved by mechanical shearing. We examined if other commercially available RNA extraction solutions are more efficient. To exclude contamination by testicular somatic cells, we prepared sperm samples using a swim-up method followed by treatment with a somatic cell lysis buffer.
This approach has previously been shown to yield sperm samples free of somatic cells Peng et al. We observed cell debris but no discernible somatic cells in the samples before RNA extraction. We assessed the quality of the extracted RNA by gel electrophoresis.
We next examined the reason for the lysis-resistance of sperm outer membrane. We tested if adding a reducing agent to AGPC solution results in the successful lysis of sperm cells. Figure 2. We then examined if the pH of the lysis solution influences the efficiency of sperm lysis.
In contrast, an acidic pH is preferred for protein disulfide bond mapping by mass spectrometry because it preserves disulfide bonds Lakbub et al. Figure 3. DTDP assay reveals pH-dependent activity of reducing agents. Tris 2-carboxyethyl phosphine TCEP was previously shown to effectively reduce disulfide bonds across a wide pH range from 1. Therefore, we first assessed if TCEP could be an effective reducing agent for acidic lysis solutions. Figure 4. As expected, complete sperm lysis improved the efficiency of RNA extraction without requiring any adjustment or additional recovery step of the standard AGPC protocol.
Therefore, all samples were DNAse treated before further use. Figure 5. Only clumped sperm tails remain visible indicating complete lysis of sperm heads. In the second row, color contrast of microscopy pictures was increased to highlight the swelling. To determine the differences in RNA yield by quantitative PCR qPCR , all sperm samples were processed similarly from lysis to final reverse transcription without any adjustment to volumes at the different processing steps.
This ensured that initial differences in RNA quantity remained until qPCR and were quantified by reporting the cycle threshold number Cq. Furthermore, we conducted end-point PCR for two less abundant genes, metastasis associated lung adenocarcinoma transcript 1 Malat1 and germ-cell specific phosphoglycerate kinase 2 Pgk2. Malat1, a long non-coding RNA expressed in many mouse tissues Hutchinson et al. In contrast, Pgk2 only amplified in sperm and testis samples Figure 6F. However, all sperm samples amplified above 40 cycles and were considered as not detectable data not shown; Bustin et al.
Figure 6. Detected cycle thresholds Cq are plotted on the y-axis and lower Cq values signify more starting material. F The germ-line specific transcript Pgk2 is only present in sperm and testis. The results indicate that a disulfide reducing agent is necessary to lyse sperm cells, suggesting that disulfide bonds confer chemical resistance to sperm cells, in mouse and potentially other species. They also strongly inhibit the enzymatic activity of ribonucleases released during cell lysis, thus prevent the degradation of RNA in solution and contribute to the acidification of the lysate Chang and Li, An acidic pH is necessary during phenol-chloroform separation where DNA and proteins move to the organic phase while RNA remains in the aqueous phase Chomczynski and Sacchi, , Despite the general applicability and potency of AGPC lysis solutions, we observed that mouse sperm cells are not effectively lysed and require the presence of pH-compatible denaturing agents.
Sperm cells are specialized cells whose functions are to maintain the paternal genome and transfer it to an oocyte to generate offspring. Histones are retained principally at genomic regions such as transcription start sites, promoters and enhancers Jung et al. Protamines have different properties to histones, and in contrast to histones, they are highly enriched in disulfide bonds.
Intra- and inter-protamine bonds mediate the packaging of DNA into a crystalline-like toroid and stabilize this structure, which requires reducing agents for its decondensation Hisano et al. The highly condensed sperm nucleus is surrounded by the acrosome which in mice, has a distinct hook-like shape for cooperative cell motility, and carries enzymes necessary for successful sperm-oocyte fusion Moore et al.
The acrosome protects the sperm nucleus from the environment of the vagina and uterus during transit to the oviduct. It is primarily composed of proteins, ceramides and sphingomyelins with very long chain carbon length 24—34 polyunsaturated fatty acids PUFA Robinson et al.
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