Table 2 RIN-values after RNA isolation with RNAeasy kit after different fixation protocols. minus 70°C Boonfix B-RLT RNAlater True cut (dry) 7.9 7.0 8.7 9.2 8.7 7.3 8.6 8.5 8.4 7.2 8.2 8.6 Blind biopsy (NaCl) 8.1 8.1 9.1 9.1 9.1 7.4 9.3 9.2 9.0 7.1 9.0 8.5 Biopsy technique RIN-values of True-cut derived RNA were slightly lower then biopsies retrieved by the Menghini technique.
The Selleck Ipatasertib difference in RIN-values was around 1 (Table 2). The effect of the solution used during the Menghini technique on RNA quality was evaluated in RNAlater preserved/RNAeasy mini kit isolated material. The use of Menghini water was compared to Menghini NaCl. Biopsies for this comparison were retrieved from surplus tissue obtained from one research BB-94 order dog, allowing both Necrostatin-1 measurements of RNA quality and quantity. The RNA yield of Menghini NaCl was more than 5 fold higher than Menghini water. The RNA
quality however was comparable (RIN-values above 8). Comparison of RNA quality obtained from biopsies of patients revealed superior quality of Menghini NaCl biopsies compared to Menghini water sampling (RIN-values up to 8.8 compared to around RIN-values of 6 resp.). Fixation time For liver tissue kept in RNAlater additional comparisons were made to reveal a possible influence of the time interval from biopsy retrieval to carry over to the preservative. Time lags of 15, 20, 25, and 30 minutes between biopsy retrieval with the Menghini NaCl method and complete enclosing of the biopsy with RNAlater did not affect RNA quality or quantity. In addition freezing of liver biopsies kept in RNAlater at minus 20°C up to 18 months did not affect RNA quality or quantity. Gene expression The optimal number of reference genes for normalization for both Menghini biopsy techniques was determined using the GeNorm program http://medgen.ugent.be/~007E;jvdesomp/genorm. The analysis was based on the following reference genes: beta-Actin, B2M, GAPDH,
GUSB, HNRPH, HPRT, RPL8, RPS19, and RPS5, as previously described . This analysis was slightly in favor for Menghini NaCl above Menghini water, since the pairwise variation (V) was lower and more stable over a wide range of reference genes (Figure 1A, B). In both situations GAPDH, RPS5 and RPS19 are amongst the most stably expressed reference genes (Figure Thiamet G 1C, D). Figure 1 Determination of the optimal number of reference genes for normalization. The GeNorm program calculates average expression stability (M) and the expression stability value by the calculation of the pair wise variation. For example V5/V6 indicates the variation in normalization factor with 5 versus 6 reference genes. A and C: Menghini NaCl. B and D: Menghini water. Histology Three different fixation protocols (included 10% neutral buffered formalin, Boonfix, and RNAlater) designed for histological studies were compared. Histological evaluation of 24 hrs formalin fixed wedge biopsies revealed normal liver histology in healthy dogs.