Michel’s Transport Fluid 500ml
Formalin fixation and paraffin embedding are standard procedures for histopathological diagnosis and allow long-term archiving of tissue samples. The cross-linking properties of formalin cause nucleic acid fragmentation and reduce the sensitivity of PCR analysis. Michel’s medium is a well-established transport medium used by dermatologists for transporting biopsies to maintain immunoreactions bound to tissue prior to direct immunofluorescence and immunoelectron microscopy. Here we report that Michel’s medium also allows short-term preservation of DNA for PCR analysis and allows amplification of amplicons larger than 1 kb. Therefore, Michel’s medium appears to be a reserve medium to perform PCR when no other samples are available.
Michel’s medium, PCR, skin biopsy, DNA
Formalin fixation and paraffin embedding (FFPE) are standard procedures for histopathological diagnosis and allow long-term archiving of tissue samples. Unfortunately, the cross-linking properties of formalin cause nucleic acid fragmentation and reduce the quality of DNA extracted from FFPE, which can result in low sensitivity of PCR testing in some samples. Therefore, the gold standard for molecular analysis remains fresh frozen tissue, which is not suitable for the storage and transport of samples. In dermatology, a commonly used alternative means of transport and storage is Michel’s means of transport.
This medium, initially described by Michel et al. in 1972, was simplified by Niedecken and Lange and is currently used by most dermatologists to transport biopsies to laboratories for the detection of tissue-bound immunoreactions by direct immunofluorescence microscopy (DIF). More recently, normal saline was shown to be an alternative to Michel’s transport medium for DIF studies, but it can only be used if samples are transported to the laboratory within 24 h. We wanted to assess whether DNA extracted from biopsies made for DIF and transported in various transport media, including Michel’s transport medium, was equally suitable for molecular testing by PCR.
Materials and methods
1. Tissue samples
All the samples came from the dermatology laboratory of the Geneva University Hospital (table (table1) .1). Twelve biopsies in Michel’s medium (55 g of NH4SO4 per 100 ml of 0.9% NaCl, pH 7.4) and 7 biopsies in 0.9% NaCl were sent for DIF by dermatologists outside the Hospital. Samples in Michel’s medium and 0.9% NaCl were sent by regular mail and reached the laboratory within 3 days of shipment. These samples were rinsed in PBS for 3 x 10 min and frozen at -20 ° C.
Twelve biopsies were from patients examined at the dermatology clinic of the Geneva University Hospital. The samples were transferred to cryotubes, snap-frozen in liquid nitrogen, and transported in liquid nitrogen to the laboratory, and immediately stored at -20 ° C. All samples were embedded in OCT and used for DIF. After sectioning, the samples were covered with OCT and archived at -20 ° C before being used for DNA extraction.
DNA was extracted and purified as previously described by Yehia et al. with minor modifications. Five to ten 10 µm cryosections from each OCT block were collected into pre-cooled tubes and lysed in 400 µl cell lysis solution (Qiagen). 30 µg of proteinase K was added to each sample. After brief vortexing, the samples were incubated overnight with shaking at 56 ° C. After a 30 min incubation at 37 ° C with RNAse A (20 μg / tube), the samples were centrifuged at 13,000 g for 5 min to remove residual debris.
The supernatant was collected and chilled on ice for 1 min, and 133 µl of protein precipitation solution (Qiagen) was added. After vortexing, the samples were left on ice for at least 20 min and then centrifuged at 13,000 g for 5 min. The supernatant containing the DNA was transferred to a fresh tube and the DNA was precipitated by the addition of 400 µl of 100% isopropanol and 1 µl of glycogen solution (20 mg/ml; Qiagen). After mixing and centrifuging at 13,000 g for 5 min, the DNA pellet was washed twice with 70% ethanol, dried, and resuspended in 30 µl of 10 mM Tris buffer, pH 8.5.
PCR was performed on genomic DNA using 35 cycles for each set of Taq DNA polymerase primers (Life Technologies) in a final volume of 25 µl. Water was used instead of a DNA template for negative controls. The β-globin (HBB) gene was amplified using primer sets GH20 (5′-GAA GAG CCA AGG ACA GGT AC-3 ′) and PCO4 (5′-CAA CTT CAT CCA CGT TCA CC-3 ′) with the following PCR conditions: 96 ° C / the 30s, 55 ° C / 30s, 72 ° C / 30s. The type I collagen gene (COL1A1) was amplified using the primer sets ex48 (5′-CCA CCT CAA GAG AAG GCT CAC GA-3 ′) and re52 (5′-TGG GAT GGA GGG AGT TTA CA-3 ′ ) with the following PCR conditions: 96 ° C / 30 s, 55 ° C / 30 s, 72 ° C / 95 s.
The extracted DNA was quantified by absorbance and evaluated for impurities by absorbance ratios at 260 nm / 280 nm (A260 / 280) for residual protein contaminants. As shown in Table 1, the DNA yield varied considerably between the different samples, probably because the cross-sectional tissue area and the number of sections used for DNA extraction were different. A mean of 14.6, 12.5, and 19.9 ng / μl of DNA were obtained for the biopsies transported in liquid nitrogen, Michel’s medium, or normal chloride, respectively.
The transport medium did not significantly influence the purity of the sample measured by the A260 / 280 proportions, with an average proportion greater than 1.8 for all of them indicating less contamination of the DNA by proteins. Amplification of small PCR products is sufficient for the diagnosis of skin infections such as syphilis, Lyme borreliosis, leishmaniasis, or tuberculosis, as well as for lymphoma. Therefore, we evaluated whether the quality of the extracted DNA was sufficient to allow PCR amplification of a 268 bp DNA fragment from the HBB gene and a 1360 bp DNA fragment from the COL1A1 gene.
In the first round of PCR, 2 µl of purified DNA was used for PCR. As shown in Table 1, most of the samples were positive for both genes, indicating that the transport medium did not significantly influence the PCR efficiency. Then, PCR reactions were performed using a similar amount of template, 40 and 8 ng, from each of the fixation methods to emphasize any difference in sample quality. As shown in Figure 1, all samples were positive with 40 and 8 ng of template.