Yoshii A, Koji T, Ohsawa N, Nakane P K
First Department of Internal Medicine, Osaka Medical College, Japan.
J Histochem Cytochem. 1995 Mar;43(3):321-7. doi: 10.1177/43.3.7532657.
Assessments of RNA integrity and its hybridizability are essential for successful implementation of in situ hybridization (ISH) for mRNA or viral RNA, particularly when paraffin-embedded specimens from surgical, biopsy, and autopsy cases are used. In this study, we examined the suitability of ISH of 28S ribosomal RNA (rRNA) for this purpose. Oligo-DNA with nucleotide sequences complementary to a well-conserved segment of 28S rRNA with auxiliary adenine-thymine-thymine (ATT) repeats at the 3' and 5' ends was synthesized. The oligo-DNA was made antigenic by converting the adjacent thymines to T-T dimers by UV irradiation and was used as a probe for ISH of 28S rRNA. The T-T dimers were detected by enzyme immunohistochemistry. When the results of ISH rRNA staining and that of total RNA staining by methyl green/pyronin Y were compared for various types of sections prepared from rat and human tissues, the staining intensities of total RNA did not always match those of ISH rRNA staining. In paraffin sections of formalin-fixed tissues, the degree of proteinase digestion influenced the ISH rRNA staining intensity, whereas it had no effect on the total RNA staining intensity. The intensities of ISH rRNA staining agreed well with those of various types of mRNA staining by ISH in 10 cases of paraffin-embedded pathological specimens. We therefore believe that ISH rRNA staining is a convenient parameter for evaluation of levels of hybridizable RNAs in tissue sections.
评估RNA完整性及其杂交能力对于成功实施针对mRNA或病毒RNA的原位杂交（ISH）至关重要，特别是当使用来自手术、活检和尸检病例的石蜡包埋标本时。在本研究中，我们为此目的检测了28S核糖体RNA（rRNA）原位杂交的适用性。合成了具有与28S rRNA保守区段互补的核苷酸序列且在3'和5'端带有辅助腺嘌呤-胸腺嘧啶-胸腺嘧啶（ATT）重复序列的寡聚DNA。通过紫外线照射将相邻的胸腺嘧啶转化为T-T二聚体，使寡聚DNA具有抗原性，并将其用作28S rRNA原位杂交的探针。通过酶免疫组织化学检测T-T二聚体。当比较从大鼠和人类组织制备的各种类型切片的rRNA原位杂交染色结果与甲基绿/派洛宁Y对总RNA的染色结果时，总RNA的染色强度并不总是与rRNA原位杂交染色强度相匹配。在福尔马林固定组织的石蜡切片中，蛋白酶消化程度影响rRNA原位杂交染色强度，而对总RNA染色强度没有影响。在10例石蜡包埋病理标本中，rRNA原位杂交染色强度与各种类型mRNA原位杂交染色强度非常一致。因此，我们认为rRNA原位杂交染色是评估组织切片中可杂交RNA水平的一个方便参数。
