Optimizing RNA quality in cryopreserved tissues without preservatives: impact of preservatives, thawing methods and...

RNA degradation during freeze‒thaw cycles in cryopreserved tissues is a major challenge for biomedical research, particularly when tissues are stored without preservatives. While agents such as TRIzol and RNALater are effective for fresh tissues, their utility for archival frozen tissues remains unclear.

We evaluated various RNA preservation strategies for frozen rabbit kidney tissues without preservatives, considering key variables such as thawing temperatures (ice vs. room temperature (RT)), preservatives (RNALater, TRIzol, and RL lysis buffers), processing delay (time before disruption), tissue aliquot sizes (ranging from 70-100 mg, 100-150 mg, to 250-300 mg), and freeze‒thaw cycles. Compared with RT-treated frozen rabbit tissues, preservative-treated tissues presented significantly greater RNA integrity when thawed on ice (p < 0.01). The RNALater group performed best in maintaining high-quality RNA (RIN ≥ 8). Although a significant difference in the RIN was observed between the 120-minute and 7-day processing delays (9.38 ± 0.10 vs. 8.45 ± 0.44), all the samples ≤ 30 mg maintained a RIN ≥ 8. For tissues ≤ 100 mg, thawing overnight on ice or at -20 °C maintained a marginally higher RIN (RIN ≥ 7). However, larger tissue aliquots (250-300 mg) presented significantly lower RINs with ice thawing than with thawing at -20 °C (5.25 ± 0.24 vs. 7.13 ± 0.69). After 3-5 freeze‒thaw cycles, tissues thawed at -20 °C presented notably greater variability in the RIN, particularly in larger tissue aliquots. In validation experiments involving cryopreserved human and murine kidney tissues, the RNALater-treated murine kidney tissues ≤ 30 mg consistently maintained high-quality RNA integrity (RIN ≥ 8), whereas the frozen human kidney tissues resulted in marginally reduced RINs compared with those of the LN grinding control (7.76 ± 0.54).

Preservatives, tissue aliquot sizes, and thawing methods significantly impact the RNA quality of frozen tissues originally stored without preservatives. Key recommendations include (1) adding RNALater during thawing, (2) thawing on ice for small aliquots (≤ 100 mg) or at -20 °C for larger samples, and (3) minimizing freeze‒thaw cycles, despite observed variations among species.