are not known can be discovered. Microarray experiments are much cheaper to perform, so large-scale studies do not typically use SAGE. Quantifying gene expressions is more exact in SAGE because it involves directly counting the number of transcripts whereas spot intensities in microarrays fall in non-discrete gradients and are prone to background noise. Variant protocols miRNA cloning MicroRNAs, or miRNAs for short, are small (~22nt) segments of RNA which have been found to play a crucial role in gene regulation. One of the most commonly used methods for cloning and identifying miRNAs within a cell or tissue was developed in the Bartel Lab and published in a paper by Lau et al. (2001). Since then, several variant protocols have arisen, but most have the same basic format. The procedure is quite similar to SAGE: The small RNA are isolated, then linkers are added to each, and the RNA is converted to cDNA by RT-PCR. Following this, the linkers, containing internal restriction sites, are digested with the appropriate restriction enzyme and the sticky ends are ligated together into concatamers. Following concatenation, the fragments are ligated into plasmids and are used to transform bacteria to generate many copies of the plasmid containing the inserts. Those may then be sequenced to identify the miRNA present, as well as analysing expression levels of a given miRNA by counting the number of times it is present, similar to SAGE. LongSAGE and RL-SAGE LongSAGE was a more robust version of the original SAGE developed in 2002 which had a higher throughput, using 20 μg of mRNA to generate a cDNA library of thousands of tags. Robust LongSage (RL-SAGE) Further improved on the LongSAGE protocol with the ability to generate a library with an insert size of 50 ng mRNA, much smaller than previous LongSAGE insert size of 2 μg mRNA and using a lower number of ditag polymerase chain reactions (PCR) to obtain a complete cDNA library. SuperSAGE SuperSAGE is a derivative of SAGE that uses the type III-endonuclease EcoP15I of phage P1, to cut 26 bp long sequence tags from each transcript's cDNA, expanding the tag-size by at least 6 bp as compared to the predecessor techniques SAGE and LongSAGE. The longer tag-size allows for a more precise allocation of the tag to the corresponding transcript, because each additional base increases the precision of the annotation considerably. Like in the original SAGE protocol, so-called ditags