Zouboulis C C, Tavakkol A
University of Michigan, Ann Arbor.
Biotechniques. 1994 Feb;16(2):290-2, 294.
The use of the storage phosphor imaging technique to quantitate radioactivity on blots generated by hybridization with 32P-cDNA probes was evaluated and compared with screen-enhanced x-ray film autoradiography. Quantitation of RNA dot blots hybridized with a 28S ribosomal RNA-specific cDNA probe showed that storage phosphor imaging was more sensitive than screen-enhanced x-ray film autoradiography in identifying low amounts of total RNA (1-10 micrograms). Evaluation of Northern blots containing 30 micrograms of total RNA from human skin biopsies hybridized with a cDNA probe for the human acidic ribosomal phosphoprotein, PO, showed that both techniques detected random biological variability of this housekeeping gene in a similar manner. The two techniques exhibited a strong linear correlation in their ability to quantitate mRNA levels of a retinoic acid-inducible gene (RIS-1). This correlation was stronger at levels corresponding to 1-fold to 30-fold increases of signal and decreased beyond this range because of the insensitivity of the x-ray film. In conclusion, storage phosphor imaging is more accurate than screen-enhanced x-ray film autoradiography in identifying different RNA amounts (higher sensitivity) and in detecting increasing RNA signals at high levels of radioactivity (higher dynamic range).
对使用存储磷光成像技术来定量通过与32P - cDNA探针杂交产生的印迹上的放射性进行了评估，并与屏幕增强型X射线胶片放射自显影进行了比较。用28S核糖体RNA特异性cDNA探针杂交的RNA斑点印迹定量分析表明，在鉴定少量总RNA（1 - 10微克）时，存储磷光成像比屏幕增强型X射线胶片放射自显影更灵敏。对含30微克来自人皮肤活检组织的总RNA且与人类酸性核糖体磷蛋白PO的cDNA探针杂交的Northern印迹进行评估，结果表明两种技术以相似的方式检测到该管家基因的随机生物学变异性。在定量视黄酸诱导基因（RIS - 1）的mRNA水平方面，这两种技术在能力上呈现出很强的线性相关性。在对应于信号增加1倍至30倍的水平时这种相关性更强，而超出此范围后相关性下降，这是因为X射线胶片的不敏感性。总之，在鉴定不同RNA量（更高灵敏度）以及在高放射性水平下检测增加的RNA信号（更高动态范围）方面，存储磷光成像比屏幕增强型X射线胶片放射自显影更准确。
